U.S. patent number 11,268,950 [Application Number 16/334,937] was granted by the patent office on 2022-03-08 for use of engineered renal tissues in assays.
This patent grant is currently assigned to Organovo, Inc.. The grantee listed for this patent is Organovo, Inc.. Invention is credited to Shelby Marie King, Deborah Lynn Greene Nguyen, Sharon C. Presnell.
United States Patent |
11,268,950 |
King , et al. |
March 8, 2022 |
Use of engineered renal tissues in assays
Abstract
Disclosed are methods of assessing the ability of a candidate
therapeutic agent to reverse, reduce or prevent renal injury by a
potential toxic agent using a three-dimensional, engineered,
bioprinted, biological renal tubule model. Also disclosed are
methods of assessing the effect of an agent on renal function, the
method comprising contacting the agent with a three-dimensional,
engineered, bioprinted, biological renal tubule model. Also
disclosed are models of renal disorder. In one embodiment,
disclosed are models of renal fibrosis, comprising a
three-dimensional, engineered, bioprinted, biological renal tubule
model. Also disclosed are methods of making the model of renal
disorder. In one embodiment disclosed are methods of making the
model of renal fibrosis comprising contacting a three-dimensional,
engineered, bioprinted, biological renal tubule model with an agent
that is capable of inducing interstitial fibrotic tissue
formation.
Inventors: |
King; Shelby Marie (San Diego,
CA), Nguyen; Deborah Lynn Greene (San Diego, CA),
Presnell; Sharon C. (San Diego, CA) |
Applicant: |
Name |
City |
State |
Country |
Type |
Organovo, Inc. |
San Diego |
CA |
US |
|
|
Assignee: |
Organovo, Inc. (San Diego,
CA)
|
Family
ID: |
1000006158454 |
Appl.
No.: |
16/334,937 |
Filed: |
September 28, 2017 |
PCT
Filed: |
September 28, 2017 |
PCT No.: |
PCT/US2017/053997 |
371(c)(1),(2),(4) Date: |
March 20, 2019 |
PCT
Pub. No.: |
WO2018/064323 |
PCT
Pub. Date: |
April 05, 2018 |
Prior Publication Data
|
|
|
|
Document
Identifier |
Publication Date |
|
US 20200018747 A1 |
Jan 16, 2020 |
|
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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62400894 |
Sep 28, 2016 |
|
|
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62453367 |
Feb 1, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N
33/573 (20130101); C12N 5/0697 (20130101); C12N
5/0062 (20130101); G01N 33/5088 (20130101); C12N
2503/04 (20130101); G01N 2800/347 (20130101) |
Current International
Class: |
G01N
33/50 (20060101); C12N 5/00 (20060101); C12N
5/071 (20100101); G01N 33/573 (20060101) |
References Cited
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WO |
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|
Primary Examiner: Cordas; Emily A
Attorney, Agent or Firm: Sterne, Kessler, Goldstein &
Fox P.L.L.C.
Claims
What is claimed is:
1. A method of assessing the ability of a candidate therapeutic
agent to reverse, reduce or prevent renal injury by a potential
toxic agent, the method comprising: (a) contacting the potential
toxic agent with a three-dimensional, engineered, bioprinted,
biological renal tubule model comprising: (i) a layer of renal
interstitial tissue, the renal interstitial tissue comprising renal
fibroblasts and endothelial cells; and (ii) a layer of renal
epithelial tissue, the renal epithelial tissue comprising renal
tubular epithelial cells, to form the three-dimensional,
engineered, biological renal tubule model; provided that the
interstitial tissue comprises an interstitial bio-ink, and the
epithelial tissue comprises an epithelial bio-ink; (b) contacting
the renal tubule model with the candidate therapeutic agent; (c)
measuring the viability or functionality of the renal tubular
epithelial cells; and (d) assessing the ability of the candidate
therapeutic agent to reverse, reduce or prevent renal injury by the
potential toxic agent based on the measured viability or
functionality of the renal tubular epithelial cells compared to a
control renal tubule model that has not been contacted with the
candidate therapeutic agent.
2. The method of claim 1, wherein the renal tubular epithelial
cells are polarized.
3. The method of claim 1, wherein the layer of renal interstitial
tissue possesses an apical and basolateral surface.
4. The method of claim 1, wherein the renal tubule model further
comprises a layer of basement membrane between the renal
interstitial tissue layer and the renal epithelial tissue
layer.
5. The method of claim 4, wherein the layer of renal epithelial
tissue is in continuous contact with the layer of basement membrane
and wherein the layer of basement membrane is in continuous contact
with the layer of renal interstitial tissue.
6. The method of claim 1, wherein the layer of renal epithelial
tissue is comprises a monolayer over 80% or more of its surface
area.
7. The method of claim 1, wherein the renal tubular epithelial
cells are the only cells present in the layer of renal epithelial
tissue and/or the fibroblasts and endothelial cells are the only
cells present in the layer of renal interstitial tissue.
8. The method of claim 1, wherein the layer of renal interstitial
tissue further comprises interstitial fibrotic tissue.
9. The method of claim 1, wherein the fibroblasts and endothelial
cells are present in the layer of renal interstitial tissue at a
ratio of about 50:50 fibroblasts to endothelial cells.
10. The method of claim 1, wherein the layer of renal interstitial
tissue or layer of renal epithelial tissue is between 70%-100%
living cells by volume.
11. The method of claim 1, wherein the renal tubule model further
comprises a biocompatible membrane.
12. The method of claim 1, wherein the renal tubule model is of
uniform thickness.
13. The method of claim 1, wherein the fibroblasts and endothelial
cells are present in a ratio at which the renal tubule model is
planar six days post-printing and prior to contacting with the
potential toxic agent.
14. The method of claim 1, wherein a plurality of the renal tubule
models are configured to form an array.
15. The method of claim 14, wherein the array is present in the
wells of a microtiter plate.
16. The method of claim 1, wherein the viability or functionality
of the renal tubular epithelial cells is determined by measuring an
indicator of metabolic activity.
17. The method of claim 16, wherein the indicator of metabolic
activity is resazurin reduction or tetrazolium salt reduction in
the renal tubule model compared to a control.
18. The method of claim 1, wherein the viability or functionality
of the renal tubular epithelial cells is measured over time.
19. The method of claim 1, which is a method to reverse or reduce
injury by the potential toxic agent, and the renal tubule model is
contacted first with the potential toxic agent and then with the
candidate therapeutic agent.
20. The method of claim 1, which is a method to reduce or prevent
injury by the potential toxic agent, and the renal tubule model is
contacted first with the candidate therapeutic agent and then with
the potential toxic agent.
Description
BACKGROUND OF THE INVENTION
Field of the Invention
The invention is in the field of renal tubule models and their use
in assays. Disclosed are methods of assessing the ability of a
candidate therapeutic agent to reverse, reduce, or prevent renal
injury by a potential toxic agent using a three-dimensional,
engineered, bioprinted, biological renal tubule model. Also
disclosed are methods of assessing the effect of an agent on renal
function, the method comprising contacting the agent with a
three-dimensional, engineered, bioprinted, biological renal tubule
model. Also disclosed are models of a renal disorder. In one
embodiment, disclosed are models of renal fibrosis, comprising a
three-dimensional, engineered, bioprinted, biological renal tubule
model. Also disclosed are methods of making the model of a renal
disorder. In one embodiment, disclosed are methods of making the
model of renal fibrosis comprising contacting a three-dimensional,
engineered, bioprinted, biological renal tubule model with an agent
that is capable of inducing interstitial fibrotic tissue
formation.
Background Art
The kidneys play a central role in the metabolism and elimination
of a variety of drugs, with the proximal tubule (PT) being exposed
to high concentrations of reactive hydrophilic metabolites at both
the luminal surface following filtration of plasma at the
glomerulus, as well as the basolateral surface following absorption
from the peritubular capillaries. Due to the action of renal
xenobiotic transporters expressed in the PT epithelium,
pharmaceutical compounds can accumulate and become concentrated in
the PT and may then undergo further metabolism by cytochrome P450
enzymes and UDP-glucuronyltransferases (Lohr et al., 1998). While
this serves a role in detoxifying these compounds to generate more
hydrophilic molecules that are secreted into the urine, highly
toxic intermediate metabolites can accumulate and cause damage to
the tubular epithelium and surrounding cells (Choudhury and Ahmed,
2006). As such, a major challenge in bringing new drugs to market
is the risk of nephrotoxicity, which is often detected late in drug
development; attrition due to nephrotoxicity accounts for 2% of
preclinical drug attrition but 19% of attrition during more costly
phase 3 clinical trials (Redfern, 2010). Post-approval,
drug-induced nephrotoxicity accounts for as much as 18-27% of cases
of acute kidney injury (AKI) (Loghman-Adham et al., 2012), with up
to 36% of these injuries related to commonly used antibiotics such
as aminoglycosides (Kleinknecht et al., 1987). While many of these
AKI cases are reversible, some drugs can induce chronic renal
injury resulting in tubular necrosis, tubulointerstitial
inflammation, and fibrosis (Kleinknecht et al., 1987; Choudhury and
Ahmed, 2006). Currently, diagnosis of AKI or renal failure relies
on elevated creatinine or blood urea nitrogen levels, which do not
become reliably clinically significant until the injury is severe
(Rahman et al., 2012). The lasting effects of AKI are significant,
with 13% of patients requiring continued dialysis and 41% of
patients requiring kidney transplant due to renal insufficiency
(Vaidya et al., 2008). Better predictive tools for identifying
nephrotoxic drugs during the drug development process would
therefore reduce the costs associated both with bringing a new drug
to market and in treating the downstream effects of AKI, as well as
improving patients' lives.
Currently, widely used screening tools for nephrotoxic compounds
consist primarily of panels of human and animal renal proximal
tubule epithelial cells (RPTEC) or small animal models. However,
these systems often fail to accurately predict organ-specific
toxicity, either as a result of species-specific differences, or
the inability to recapitulate relevant aspects of kidney
physiology, including toxicity following xenobiotic transport and
biotransformation (Lin and Will, 2012). While freshly isolated
primary human RPTEC obviate differences in species specificity, the
cells rapidly dedifferentiate and senesce when cultured in
isolation, losing expression of key transporters and metabolic
enzymes (Wieser et al., 2008; Vesey et al., 2009). In the human
kidney, the RPTEC exist in close connection with the renal
interstitium, defined as the space between the cortical tubules
comprising cells, extracellular matrix, proteoglycans,
glycoproteins, and interstitial fluid (Lemley and Kriz, 1991). The
cell types found in the cortical interstitium include
fibroblast-like cells and immune cells, which are interspersed with
the microvasculature of peritubular capillaries (Brenner, 2008).
These supporting cell types may play a key role in maintaining the
continued function of RPTEC, as co-culture of primary RPTEC with
endothelial cells results in a robust paracrine signaling network
that improves RPTEC proliferation and differentiation (Tasnim and
Zink, 2012). Thus, placing primary RPTEC together with supporting
interstitial cells in a more native, three-dimensional (3D)
architecture may aid in maintaining their function over time, as
well as allowing for assessment of additional types of kidney
injury that are difficult to model using epithelial cells alone,
such as fibrosis (Subramanian et al., 2010).
One of the primary aims of tissue engineering is to use living
cells and biomaterials to generate 3D tissues that recapitulate key
aspects of the architecture and function of a native tissue or
organ. With proper in vitro or in vivo conditioning, the cells
within these structures can respond to soluble and mechanical cues
by establishing cell-cell and cell-matrix interactions that mimic
some aspects of native tissue (Griffith et al., 2014). It is well
established that cells cultured in 3D configurations, such as
spheroids or collagen gels, perform differently in functional
assays than 2D cultures, and the physiologic responses of cells in
3D more closely approximate responses observed in vivo (Godoy et
al., 2013). One such means for fabricating these 3D structures is
bioprinting. In this approach, bioinks composed of cellular
material are extruded in reproducible, geometrically-defined
patterns created by the investigator (Ozbolat and Hospodiuk, 2016).
The bioink is composed of self-assembling multicellular aggregates
that adhere to one another following deposition, leading to
formation of complex, patterned tissues (Jakab et al., 2008; Jakab
et al., 2010). Combining the use of self-assembling multicellular
aggregates with computer-controlled bioprinting allows the creation
of highly reproducible, scaffold-free tissues that form and mature
in the absence of exogenous extracellular matrix that can interfere
with direct cell-cell contacts (Norotte et al., 2009).
SUMMARY OF THE INVENTION
Bioprinting technology was leveraged to design and create layered
tissue models of the human PT that incorporate key interstitial
cell types supporting RPTEC to facilitate both cell-cell
interactions and paracrine signaling between renal fibroblasts,
endothelial cells, and epithelial cells. The resulting tissues
supported epithelial morphology and function for at least 30 days
in culture, and were effectively used to model the role of the
organic cation transporter OCT2 in nephrotoxic responses to
cisplatin using a combination of biochemical, transcriptional, and
histological endpoints. Thus, this system is useful in predicting
nephrotoxicity of pharmaceutical compounds earlier in the drug
development process.
The engineered tissues described herein represent a model of the
tubulointerstitial interface in which human renal interstitial
tissue is supporting human renal proximal tubule epithelial cells
to facilitate their optimal morphology and function. Creation of a
three-dimensional tubulointerstitial interface facilitates the
correct localization of drug transporters and receptors required
for metabolism in order to accurately study how small molecules,
chemicals, contaminants, or biologics affect the renal proximal
tubule. This represents a more physiologically relevant alternative
to two-dimensional monolayers of human or canine kidney epithelial
cells and serves as an adjunct to, or in some cases, replacement of
animal studies in which species difference in renal functions
hamper interpretation of results.
The engineered tissues described herein provide an opportunity to
accurately study how compounds affect the renal proximal tubule as
well as modeling pathogenic processes that involve tubular
transport, cell-cell interactions, and the development of renal
disorders such as may occur in chronic renal disease, polycystic
kidney disease, or type II diabetes.
The engineered tissues described herein provide an opportunity to
accurately study how compounds affect the renal proximal tubule as
well as modeling pathogenic processes that involve tubular
transport, cell-cell interactions, and the development of renal
disorders such as may occur in chronic renal disease, polycystic
kidney disease, or type II diabetes.
The engineered tissues described herein provide an opportunity to
accurately study how compounds affect the renal proximal tubule as
well as modeling pathogenic processes that involve tubular
transport, cell-cell interactions, and the development of
tubulointerstitial fibrosis such as may occur in chronic renal
disease, polycystic kidney disease, or type II diabetes.
Provided are methods of assessing the ability of a candidate
therapeutic agent to reverse, reduce, or prevent renal injury by a
potential toxic agent, the method comprising: contacting the
potential toxic agent with a three-dimensional, engineered,
bioprinted, biological renal tubule model; contacting the renal
tubule model with the candidate therapeutic agent; determining the
viability or functionality of the renal tubular epithelial cells;
and assessing the ability of the candidate therapeutic agent to
reverse, reduce, or prevent renal injury by the potential toxic
agent based on the determined viability or functionality of the
renal tubular epithelial cells compared to a control renal tubule
model that has not been contacted with the candidate therapeutic
agent. In some embodiments, the three-dimensional, engineered,
bioprinted, biological renal tubule model comprises a layer of
renal interstitial tissue, the renal interstitial tissue comprising
renal fibroblasts and endothelial cells; and a layer of renal
epithelial tissue, the renal epithelial tissue comprising renal
tubular epithelial cells; provided that the interstitial tissue
comprises an interstitial bio-ink, the epithelial tissue comprises
an epithelial bio-ink, and form a three-dimensional, engineered,
biological renal tubule model.
In some embodiments, the renal tubular epithelial cells are
polarized. In some embodiments, the layer of renal interstitial
tissue possesses an apical and basolateral surface.
In some embodiments, the model further comprises a layer of
basement membrane between the renal interstitial tissue layer and
the renal epithelial tissue layer. In some embodiments, the layer
of renal epithelial tissue is in continuous contact with the layer
of basement membrane, and the layer of basement membrane is in
continuous contact with the layer of renal interstitial tissue.
In some embodiments, the layer of renal epithelial tissue is
substantially a monolayer.
In some embodiments, the renal tubular epithelial cells are the
only cells present in the layer of renal epithelial tissue. In
other embodiments, the fibroblasts and endothelial cells are the
only cells present in the layer of renal interstitial tissue. In
other embodiments, the layer of renal interstitial tissue further
comprises interstitial fibrotic tissue.
In some embodiments, the fibroblasts and endothelial cells are
present in the layer of renal interstitial tissue at a ratio of
about 50:50 fibroblasts to endothelial cells. In some embodiments,
the layer of renal interstitial tissue or layer of renal epithelial
tissue is between 70%-100% living cells by volume. In other
embodiments, the layer of renal interstitial tissue further
comprises interstitial fibrotic tissue.
In some embodiments, the renal tubule model further comprises a
biocompatible membrane.
In some embodiments, the renal tubular model is of uniform
thickness. In some embodiments, the renal tubular model is at least
2 cell layers thick.
In some embodiments, the fibroblasts and endothelial cells are
present in a ratio at which the renal tubule model is planar six
days post-printing.
In some embodiments, a plurality of the renal tubule models are
configured to form an array. In some embodiments, the array is
present in the wells of a microtiter plate.
In some embodiments, the potential toxic agent is a toxin, a
therapeutic agent, an antimicrobial agent, a metal, or an
environmental agent.
In other embodiments, the potential toxic agent is an antiviral, an
analgesic agent, an antidepressant agent, a diuretic agent, or a
proton pump inhibitor.
In other embodiments, the potential toxic agent is a cytokine, a
chemokine, a small molecule drug, a large molecule drug, a protein
or a peptide.
In other embodiments, the potential toxic agent is a
chemotherapeutic agent which is an aromatase inhibitor; an
anti-estrogen; an anti-androgen; a gonadorelin agonist; a
topoisomerase I inhibitor; a topoisomerase II inhibitor; a
microtubule active agent; an alkylating agent; a retinoid, a
carontenoid, or a tocopherol; a cyclooxygenase inhibitor; an MMP
inhibitor; an mTOR inhibitor; an antimetabolite; a platin compound;
a methionine aminopeptidase inhibitor; a bisphosphonate; an
antiproliferative antibody; a heparanase inhibitor; an inhibitor of
Ras oncogenic isoforms; a telomerase inhibitor; a proteasome
inhibitor; a compound used in the treatment of hematologic
malignancies; a Flt-3 inhibitor; an Hsp90 inhibitor; a kinesin
spindle protein inhibitor; a MEK inhibitor; an antitumor
antibiotic; a nitrosourea; a compound targeting/decreasing protein
or lipid kinase activity, a compound targeting/decreasing protein
or lipid phosphatase activity, or an anti-angiogenic compound.
In other embodiments, the potential toxic agent is a
chemotherapeutic agent which is daunorubicin, adriamycin, Ara-C,
VP-16, teniposide, mitoxantrone, idarubicin, cisplatin,
carboplatinum, PKC412, 6-mercaptopurine (6-MP), fludarabine
phosphate, octreotide, SOM230, FTY720, 6-thioguanine, cladribine,
6-mercaptopurine, pentostatin, hydroxyurea,
2-hydroxy-1H-isoindole-1,3-dione derivatives,
l-(4-chloroanilino)-4-(4-pyridylmethyl)phthalazine,
1-(4-chloroanilino)-4-(4-pyridylmethyl)phthalazine succinate,
angiostatin, endostatin, anthranilic acid amides, ZD4190, ZD6474,
SU5416, SU6668, bevacizumab, rhuMAb, rhuFab, macugon, FLT-4
inhibitors, FLT-3 inhibitors, VEGFR-2 IgGI antibody, RPI 4610,
bevacizumab, porfimer sodium, anecortave, triamcinolone,
hydrocortisone, 11-.alpha.-epihydrocotisone, cortex olone,
17a-hydroxyprogesterone, corticosterone, desoxycorticosterone,
testosterone, estrone, dexamethasone, fluocinolone, a plant
alkaloid, a hormonal compound and/or antagonist, a biological
response modifier, such as a lymphokine or interferon, an antisense
oligonucleotide or oligonucleotide derivative, shRNA, siRNA, or a
pharmaceutically acceptable salt thereof.
In other embodiments, the potential toxic agent is acetaminophen,
lithium, acyclovir, amphotericin B, and aminoglycoside, a beta
lactams, foscavir, ganciclovir, pentamidine, a quinolone, a
sulfonamide, vancomycin, rifampin, adefovir, indinavir, didofovir,
tenofovir, methotrexate, lansoprazole, omeprazole, pantopraxole,
allopurinol, phenytoin, ifosfamide, gentamycin, or zoledronate.
In other embodiments, the potential toxic agent is radiation.
In some embodiments, the viability or functionality of the renal
tubular epithelial cells is determined by measuring an indicator of
metabolic activity. In some embodiments, the indicator of metabolic
activity is resazurin reduction or tetrazolium salt reduction in
the renal tubule mode compared to a control.
In other embodiments, the viability or functionality of the renal
tubular epithelial cells is determined by measuring lactate
dehydrogenase (LDH) activity, gamma glutamyl-transferase (GGT)
activity, protease activity, ATP utilization, glucose uptake
activity, sodium-glucose co-transporter-2 (SGLT2) activity or RNA
expression compared to a control.
In other embodiments, the viability or functionality of the renal
tubular epithelial cells is determined by measuring a renal
transport molecule activity in the model compared to a control. In
some embodiments, the transport molecule activity is excretion
and/or uptake of at least one macromolecule. In some embodiments,
the macromolecule is albumin.
In other embodiments, the viability or functionality of the renal
tubular epithelial cells is determined by identifying regeneration
of the renal tubular epithelial cells compared to a control.
In other embodiments, the viability or functionality of the renal
tubular epithelial cells is determined by measuring the
trans-epithelial electrical resistance or the passive permeability
of the renal tubule model compared to a control.
In other embodiments, the viability or functionality of the renal
tubular epithelial cells is determined by measuring changes in
vitamin D production, changes in angiotensin conversion,
alterations to ion exchange, alterations to pH, alterations to
acid/base balance, alterations to renal tubule barrier function, or
alterations to the intrarenal renin/angiotensin system (RAS),
alterations in physiology, alterations in pathology, alterations to
transport of molecules, alterations to sodium-glucose
cotransporter-2 (SGLT2) activity, amounts of interstitial fibrotic
tissue, or regeneration of the renal tubule model compared to a
control.
In other embodiments, the viability or functionality of the renal
tubular epithelial cells is determined by measuring amounts of
interstitial fibrotic tissue compared to a control.
In other embodiments, the viability or functionality of the renal
tubular epithelial cells is measured over time.
In some embodiments, the renal tubule model is contacted first with
the potential toxic agent and then with the candidate therapeutic
agent. In other embodiments, the renal tubule model is contacted
first with the candidate therapeutic agent and then with the
potential toxic agent.
In some embodiments, the renal tubule model has been cultured in a
cell culture medium prior to being contacted with the candidate
therapeutic agent and the potential toxic agent.
In some embodiments, the renal tubule model has been cultured for 3
or more days in the cell culture medium.
Also provided are methods of assessing the effect of an agent on
renal function, the method comprising contacting the agent with a
three-dimensional, engineered, bioprinted, biological renal tubule
model; and measuring the effect of the agent on the viability or
functionality of the renal tubular epithelial cells. In some
embodiments, the three-dimensional, engineered, bioprinted,
biological renal tubule model comprises a layer of renal
interstitial tissue, the renal interstitial tissue comprising renal
fibroblasts and endothelial cells; and a layer of renal epithelial
tissue, the renal epithelial tissue comprising renal tubular
epithelial cells, to form the three-dimensional, engineered,
biological renal tubule model; provided that the interstitial
tissue comprises an interstitial bio-ink, the epithelial tissue
comprises an epithelial bio-ink, and form a three-dimensional,
engineered, biological renal tubule model.
In some embodiments, the fibroblasts and endothelial cells are
present in a ratio of fibroblasts to endothelial cells at which the
renal tubule model is planar six days post-printing.
In some embodiments, the renal tubular epithelial cells are
polarized. In some embodiments, the layer of renal interstitial
tissue possesses an apical and basolateral surface.
In some embodiments, the model further comprises a layer of
basement membrane between the renal interstitial tissue layer and
the renal epithelial tissue layer. In some embodiments, the layer
of renal epithelial tissue is in continuous contact with the layer
of basement membrane and wherein the layer of basement membrane is
in continuous contact with the layer of renal interstitial
tissue.
In some embodiments, the layer of renal epithelial tissue is
substantially a monolayer.
In some embodiments, renal tubular epithelial cells are the only
cells present in the layer of renal epithelial tissue. In other
emobodiments, the fibroblasts and endothelial cells are the only
cells present in the layer of renal interstitial tissue. In some
embodiments, the layer of renal interstitial tissue further
comprises interstitial fibrotic tissue.
In some embodiments, the fibroblasts and endothelial cells are
present in the layer of renal interstitial tissue at a ratio of
about 50:50 fibroblasts to endothelial cells.
In some embodiments, any of the layer of renal interstitial tissue
or layer of renal epithelial tissue is between 70%-100% living
cells by volume.
In some embodiments, the renal tubule model further comprises a
biocompatible membrane.
In some embodiments, the renal tubular model is of uniform
thickness. In other embodiments, the renal tubule model is 2 or
more cell layers thick.
In some embodiments, a plurality of the renal tubule models are
configured to form an array. In some embodiments, the array is
present in the wells of a microtiter plate.
In some embodiments, the agent is a toxin, a therapeutic agent, an
antimicrobial agent, a metal, or an environmental agent.
In other embodiments, the agent is an antiviral, an analgesic
agent, an antidepressant agent, a diuretic agent, or a proton pump
inhibitor.
In other embodiments, the agent is a cytokine, a chemokine, a small
molecule drug, a large molecule drug, a protein or a peptide.
In other embodiments, the agent is a chemotherapeutic agent which
is an aromatase inhibitor; an anti-estrogen; an anti-androgen; a
gonadorelin agonist; a topoisomerase I inhibitor; a topoisomerase
II inhibitor; a microtubule active agent; an alkylating agent; a
retinoid, a carontenoid, or a tocopherol; a cyclooxygenase
inhibitor; an MMP inhibitor; an mTOR inhibitor; an antimetabolite;
a platin compound; a methionine aminopeptidase inhibitor; a
bisphosphonate; an antiproliferative antibody; a heparanase
inhibitor; an inhibitor of Ras oncogenic isoforms; a telomerase
inhibitor; a proteasome inhibitor; a compound used in the treatment
of hematologic malignancies; a Flt-3 inhibitor; an Hsp90 inhibitor;
a kinesin spindle protein inhibitor; a MEK inhibitor; an antitumor
antibiotic; a nitrosourea; a compound targeting/decreasing protein
or lipid kinase activity, a compound targeting/decreasing protein
or lipid phosphatase activity, or an anti-angiogenic compound.
In other embodiments, the agent is a chemotherapeutic agent which
is daunorubicin, adriamycin, Ara-C, VP-16, teniposide,
mitoxantrone, idarubicin, cisplatin, carboplatinum, PKC412,
6-mercaptopurine (6-MP), fludarabine phosphate, octreotide, SOM230,
FTY720, 6-thioguanine, cladribine, 6-mercaptopurine, pentostatin,
hydroxyurea, 2-hydroxy-1H-isoindole-1,3-dione derivatives,
1-(4-chloroanilino)-4-(4-pyridylmethyl)phthalazine,
1-(4-chloroanilino)-4-(4-pyridylmethyl)phthalazine succinate,
angiostatin, endostatin, anthranilic acid amides, ZD4190, ZD6474,
SU5416, SU6668, bevacizumab, rhuMAb, rhuFab, macugon, FLT-4
inhibitors, FLT-3 inhibitors, VEGFR-2 IgGI antibody, RPI 4610,
bevacizumab, porfimer sodium, anecortave, triamcinolone,
hydrocortisone, 11-.alpha.-epihydrocotisone, cortex olone,
17a-hydroxyprogesterone, corticosterone, desoxycorticosterone,
testosterone, estrone, dexamethasone, fluocinolone, a plant
alkaloid, a hormonal compound and/or antagonist, a biological
response modifier, such as a lymphokine or interferon, an antisense
oligonucleotide or oligonucleotide derivative, shRNA, siRNA, or a
pharmaceutically acceptable salt thereof.
In other embodiments, the agent is acetaminophen, lithium,
acyclovir, amphotericin B, an aminoglycoside, a beta lactam,
foscavir, ganciclovir, pentamidine, a quinolone, a sulfonamide,
vancomycin, rifampin, adefovir, indinavir, didofovir, tenofovir,
methotrexate, lansoprazole, omeprazole, pantopraxole, allopurinol,
phenytoin, ifosfamide, gentamycin, or zoledronate.
In other embodiments, the agent is radiation.
In some embodiments, the viability or functionality of the renal
tubular epithelial cells is determined by measuring an indicator of
metabolic activity. In some embodiments, the indicator of metabolic
activity is resazurin reduction or tetrazolium salt reduction in
the renal tubule mode compared to a control.
In other embodiments, the viability or functionality of the renal
tubular epithelial cells is determined by measuring lactate
dehydrogenase (LDH) activity, gamma glutamyl-transferase (GGT)
activity, protease activity, ATP utilization, SGLT2 activity,
glucose uptake activity or RNA expression compared to a
control.
In other embodiments, the viability or functionality of the renal
tubular epithelial cells is determined by measuring a renal
transport molecule activity in the model compared to a control. In
some embodiments, the transport molecule activity is excretion
and/or uptake of at least one macromolecule. In some embodiments,
the macromolecule is albumin.
In other embodiments, the viability or functionality of the renal
tubular epithelial cells is determined by identifying regeneration
of the renal tubular epithelial cells compared to a control.
In other embodiments, the viability or functionality of the renal
tubular epithelial cells is determined by measuring the
trans-epithelial electrical resistance or the passive permeability
of the renal tubule model compared to a control.
In other embodiments, the viability or functionality of the renal
tubular epithelial cells is determined by measuring changes in
vitamin D production, changes in angiotensin conversion,
alterations to ion exchange, alterations to pH, alterations to
acid/base balance, alterations to renal tubule barrier function,
alterations to the intrarenal renin/angiotensin system (RAS),
alterations in physiology, alterations in pathology, alterations to
transport of molecules, alterations to sodium-glucose
cotransporter-2 (SGLT2) activity, amounts of interstitial fibrotic
tissue, or regeneration of the renal tubule model compared to a
control.
In other embodiments, the viability or functionality of the renal
tubular epithelial cells is measured over time.
In some embodiments, the renal tubule model has been cultured in a
cell culture medium prior to being contacted with the agent. In
some embodiments, the renal tubule model has been cultured for 3 or
more days in the cell culture medium.
In some embodiments, the agent is removed, and the renal tubule
model is assessed to determine whether the absence of the agent
results in improved viability or functionality of the renal tubular
epithelial cells.
Also provided are models of renal fibrosis, comprising a
three-dimensional, engineered, bioprinted, biological renal tubule
model. The three-dimensional, engineered, bioprinted, biological
renal tubule model comprises a layer of renal interstitial tissue,
the renal interstitial tissue comprising renal fibroblasts,
endothelial cells and fibrotic tissue; and a layer of renal
epithelial tissue, the renal epithelial tissue comprising renal
tubular epithelial cells, to form the three-dimensional,
engineered, biological renal tubule model; provided that the
interstitial tissue comprises an interstitial bio-ink, the
epithelial tissue comprises an epithelial bio-ink, and form a
three-dimensional, engineered, biological renal tubule model. In
some embodiments, the model displays contraction, curling,
expansion of the tissue, or another fibrosis phenotype when
fibrosis is present in the model.
In some embodiments, the renal tubular epithelial cells are
polarized. In some embodiments, the layer of renal interstitial
tissue possesses an apical and basolateral surface.
In some embodiments, the model further comprises a layer of
basement membrane between the renal interstitial tissue layer and
the renal epithelial tissue layer. In some embodiments, the layer
of renal epithelial tissue is in continuous contact with the layer
of basement membrane, and the layer of basement membrane is in
continuous contact with the layer of renal interstitial tissue.
In some embodiments, the layer of renal epithelial tissue is
substantially a monolayer.
In some embodiments, renal tubular epithelial cells are the only
cells present in the layer of renal epithelial tissue. In some
embodiments, the fibroblasts and endothelial cells are the only
cells present in the layer of renal interstitial tissue.
In some embodiments, the fibroblasts and endothelial cells are
present in the layer of renal interstitial tissue at a ratio of
about 50:50 fibroblasts to endothelial cells.
In some embodiments, the layer of renal interstitial tissue or
layer of renal epithelial tissue is between 70%-100% living cells
by volume.
In some embodiments, the renal tubule model further comprises a
biocompatible membrane.
In some embodiments, the renal tubular model displays deformation
of the planar tissue structure and excess extracellular matrix
deposition.
In some embodiments, the renal tubular model is 2 or more cell
layers thick.
In some embodiments, a plurality of the renal tubule models are
configured to form an array. In some embodiments, the array is
present in the wells of a microtiter plate.
In some embodiments, the fibroblasts and endothelial cells are
present at a ratio at which the renal tubule model is planar six
days post printing.
Also provided are methods of making the model of renal fibrosis
comprising contacting a three-dimensional, engineered, bioprinted,
biological renal tubule model with an agent that is capable of
inducing interstitial fibrotic tissue formation, wherein the renal
tubule model comprises a layer of renal interstitial tissue, the
renal interstitial tissue comprising renal fibroblasts and
endothelial cells; and a layer of renal epithelial tissue, the
renal epithelial tissue comprising renal tubular epithelial cells,
to form the three-dimensional, engineered, biological renal tubule
model; provided that the interstitial tissue comprises an
interstitial bio-ink, the epithelial tissue comprises an epithelial
bio-ink, and form a three-dimensional, engineered, biological renal
tubule model.
In some embodiments, the fibroblasts and endothelial cells are
present in a ratio of fibroblasts to endothelial cells at which the
renal tubule model is planar six days post printing.
In some embodiments, the agent that is capable of inducing
interstitial fibrotic tissue deposition is cyclosporine A,
aristolochoic acid, tacrolimus, TGFbeta, cisplatin, acyclovir,
allopurinol, beta lactam antibiotics, indinavir, lansoprazole,
omeprazole, pantoprazole, phenytoin, ranitidine, or vancomycin.
Also provided is a model of a renal disorder, comprising a
three-dimensional, engineered, bioprinted, biological tubule model
comprising: (a) a layer of renal interstitial tissue, the renal
interstitial tissue comprising renal fibroblasts and endothelial
cells; and (b) a layer of renal epithelial tissue, the renal
epithelial tissue comprising renal tubular epithelial cells, to
form the three-dimensional, engineered, biological renal tubule
model; provided that the interstitial tissue comprises an
interstitial bio-ink, the epithelial tissue comprises an epithelial
bio-ink, and form a three-dimensional, engineered, biological renal
tubule model, wherein the model comprises a phenotype that is
characteristic of a renal disorder in the renal tubule.
In some embodiments, the phenotype includes at least one of
contraction, curling, expansion, necrosis, apoptosis, tubular
regeneration, compensatory proliferation, epithelial-mesenchymal
transition, inflammation, ischemia, ischemia/reperfusion, reactive
oxygen species, changes in the mitochondria, changes to cell
morphology, changes to nuclear morphology, hyperproliferation,
alterations in gene expression, secretion of biomarkers, epigenetic
modifications, crystal deposition, cyst formation, a change to a
cellular function, angiogenesis, hypoxia, extraceullar matrix
deposition, or death of surrounding tissue.
In some embodiments, the renal tubular epithelial cells are
polarized.
In some embodiments, the layer of renal interstitial tissue
possesses an apical and basolateral surface.
In some embodiments, the model further comprises a layer of
basement membrane between the renal interstitial tissue layer and
the renal epithelial tissue layer.
In some embodiments, the layer of renal epithelial tissue is in
continuous contact with the layer of basement membrane and wherein
the layer of basement membrane is in continuous contact with the
layer of renal interstitial tissue.
In some embodiments, the layer of renal epithelial tissue is
substantially a monolayer.
In some embodiments, renal tubular epithelial cells are the only
cells present in the layer of renal epithelial tissue.
In some embodiments, the fibroblasts and endothelial cells are the
only cells present in the layer of renal interstitial tissue.
In some embodiments, the fibroblasts and endothelial cells are
present in the layer of renal interstitial tissue at a ratio of
about 50:50 fibroblasts to endothelial cells.
In some embodiments, the layer of renal interstitial tissue or
layer of renal epithelial tissue is between 70%-100% living cells
by volume.
In some embodiments, the renal tubule model further comprises a
biocompatible membrane.
In some embodiments, the renal tubular model displays deformation
of the planar tissue structure and excess extracellular matrix
deposition.
In some embodiments, the renal tubular model is at least 2 cell
layers thick.
In some embodiments, a plurality of the renal tubule models are
configured to form an array.
In some embodiments, the array is present in the wells of a
microtiter plate.
In some embodiments, the fibroblasts and endothelial cells are
present at a ratio at which the renal tubule model is planar six
days post printing.
In some embodiments, the renal disorder is associated with a
congenital abnormality, diabetes, an immune complex disease,
vascular sclerosis, renal ablation, renal fibrosis, hypertension,
arterionephrosclerosis, lupus nephritis, vascular disease,
inflammation, hemolytic-uremic syndrome, obstructive nephropathy,
dyslipoproteinemia, recurrent dehydration, reflux nephropathy,
radiation nephropathy, atheroembolic renal disease, scleroderma,
sickle cell anemia, retention of lipids, infection, ischemia,
ischemia/reperfusion, a transport deficiency, crystal deposition, a
genetic disorder, a chronic system disorder, renal cancer, or a
combination thereof.
In some embodiments, the phenotype is induced by contacting the
renal tubule model with a treatment, compound, or infectious agent
that gives rise to the phenotype.
In some embodiments, the phenotype is the presence of a tumor, a
tumor fragment, a tumor cell, or an immortalized cell in the the
renal tubule model.
In some embodiments, the fibroblasts, endothelial cells, epithelial
cells, or combinations thereof of the renal tubule model are
primary cells obtained from a diseased donor.
In some embodiments, further comprising a genetically modified
cell, wherein the phenotype is induced by the genetically modified
cell.
In some embodiments, the treatment, compound, or infectious agent
that gives rise to the phenotype is a toxin, a potential toxic
agent, an antimicrobial agent, a metal, or an environmental
agent.
In some embodiments, the potential toxic agent is an
anti-infective, an analgesic agent, an antidepressant agent, a
diuretic agent, or a proton pump inhibitor.
In some embodiments, the potential toxic agent is a cytokine, a
chemokine, a small molecule drug, a large molecule drug, a protein,
or a peptide.
In some embodiments, the potential toxic agent is a
chemotherapeutic agent which is an aromatase inhibitor; an
anti-estrogen; an anti-androgen; a gonadorelin agonist; a
topoisomerase I inhibitor; a topoisomerase II inhibitor; a
microtubule active agent; an alkylating agent; a retinoid, a
carontenoid, or a tocopherol; a cyclooxygenase inhibitor; an MMP
inhibitor; an mTOR inhibitor; an antimetabolite; a platin compound;
a methionine aminopeptidase inhibitor; a bisphosphonate; an
antiproliferative antibody; a heparanase inhibitor; an inhibitor of
Ras oncogenic isoforms; a telomerase inhibitor; a proteasome
inhibitor; a compound used in the treatment of hematologic
malignancies; a Flt-3 inhibitor; an Hsp90 inhibitor; a kinesin
spindle protein inhibitor; a MEK inhibitor; an antitumor
antibiotic; a nitrosourea; a compound targeting/decreasing protein
or lipid kinase activity; a compound targeting/decreasing protein
or lipid phosphatase activity; or an anti-angiogenic compound.
In some embodiments, the potential toxic agent is a
chemotherapeutic agent which is daunorubicin, adriamycin, Ara-C,
VP-16, teniposide, mitoxantrone, idarubicin, cisplatin,
carboplatinum, PKC412, 6-mercaptopurine (6-MP), fludarabine
phosphate, octreotide, SOM230, FTY720, 6-thioguanine, cladribine,
6-mercaptopurine, pentostatin, hydroxyurea,
2-hydroxy-1H-isoindole-1,3-dione derivatives,
l-(4-chloroanilino)-4-(4-pyridylmethyl)phthalazine,
1-(4-chloroanilino)-4-(4-pyridylmethyl)phthalazine succinate,
angiostatin, endostatin, anthranilic acid amides, ZD4190, ZD6474,
SU5416, SU6668, bevacizumab, rhuMAb, rhuFab, macugon, FLT-4
inhibitors, FLT-3 inhibitors, VEGFR-26 IgGI antibody, RPI 4610,
bevacizumab, porfimer sodium, anecortave, triamcinolone,
hydrocortisone, 11-.alpha.-epihydrocotisone, cortex olone,
17a-hydroxyprogesterone, corticosterone, desoxycorticosterone,
testosterone, estrone, dexamethasone, fluocinolone, a plant
alkaloid, a hormonal compound and/or antagonist, a biological
response modifier, such as a lymphokine or interferon, an antisense
oligonucleotide or oligonucleotide derivative, shRNA, siRNA, or a
pharmaceutically acceptable salt thereof.
In some embodiments, the potential toxic agent is acetaminophen,
lithium, acyclovir, amphotericin B, an aminoglycoside, a beta
lactam, foscavir, ganciclovir, pentamidine, a quinolone, a
sulfonamide, vancomycin, rifampin, adefovir, indinavir, didofovir,
tenofovir, methotrexate, lansoprazole, omeprazole, pantopraxole,
allopurinol, phenytoin, ifosfamide, gentamycin, or zoledronate.
In some embodiments, the potential toxic agent is radiation.
In some embodiments, the renal disorder is acute renal disorder,
chronic renal disorder, or renal cancer.
Also provided is a method of assessing the ability of a candidate
therapeutic agent to reverse, reduce, induce, or prevent a renal
disorder, the method comprising: (a) contacting a renal tubule
model with a candidate therapeutic agent; (b) determining a
viability or functionality of the renal tissue cells; and (c)
assessing an ability of the candidate therapeutic to reverse,
reduce, induce, or prevent a renal disorder based on the determined
viability or functionality of the renal tissue cells compared to a
control renal tubule model that has not been contacted with the
candidate therapeutic agent.
In some embodiments, the method of assessing a candidate
therapeutic further comprises: (d) removing the candidate
therapeutic agent; and (e) assessing whether the absence of the
agent results in improved viability or functionality of the renal
tissue cells.
In some embodiments, the phenotype is the presence of a tumor, a
tumor fragment, a tumor cell, or an immortalized cell in the the
renal tubule model.
In some embodiments, the fibroblasts, endothelial cells, epithelial
cells, or combinations thereof of the renal tubule model are
primary cells obtained from a diseased donor.
In some embodiments, the candidate therapeutic agent is an
antiviral, an analgesic agent, an antidepressant agent, a diuretic
agent, or a proton pump inhibitor.
In some embodiments, the candidate therapeutic agent is a cytokine,
a chemokine, a small molecule drug, a large molecule drug, a
protein, or a peptide.
In some embodiments, the candidate therapeutic agent is a
chemotherapeutic agent which is an aromatase inhibitor; an
anti-estrogen; an anti-androgen; a gonadorelin agonist; a
topoisomerase I inhibitor; a topoisomerase II inhibitor; a
microtubule active agent; an alkylating agent; a retinoid, a
carontenoid, or a tocopherol; a cyclooxygenase inhibitor; an MMP
inhibitor; an mTOR inhibitor; an antimetabolite; a platin compound;
a methionine aminopeptidase inhibitor; a bisphosphonate; an
antiproliferative antibody; a heparanase inhibitor; an inhibitor of
Ras oncogenic isoforms; a telomerase inhibitor; a proteasome
inhibitor; a compound used in the treatment of hematologic
malignancies; a Flt-3 inhibitor; an Hsp90 inhibitor; a kinesin
spindle protein inhibitor; a MEK inhibitor; an antitumor
antibiotic; a nitrosourea; a compound targeting/decreasing protein
or lipid kinase activity; a compound targeting/decreasing protein
or lipid phosphatase activity; or an anti-angiogenic compound.
In some embodiments, the candidate therapeutic agent is a
chemotherapeutic agent which is daunorubicin, adriamycin, Ara-C,
VP-16, teniposide, mitoxantrone, idarubicin, cisplatin,
carboplatinum, PKC412, 6-mercaptopurine (6-MP), fludarabine
phosphate, octreotide, SOM230, FTY720, 6-thioguanine, cladribine,
6-mercaptopurine, pentostatin, hydroxyurea,
2-hydroxy-1H-isoindole-1,3-dione derivatives,
1-(4-chloroanilino)-4-(4-pyridylmethyl)phthalazine,
1-(4-chloroanilino)-4-(4-pyridylmethyl(phthalazine succinate,
angiostatin, endostatin, anthranilic acid amides, ZD4190, ZD6474,
SU5416, SU6668, bevacizumab, rhuMAb, rhuFab, macugon, FLT-4
inhibitors, FLT-3 inhibitors, VEGFR-2 IgGI antibody, RPI 4610,
bevacizumab, porfimer sodium, anecortave, triamcinolone,
hydrocortisone, 11-.alpha.-epihydrocotisone, cortex olone,
17a-hydroxyprogesterone, corticosterone, desoxycorticosterone,
testosterone, estrone, dexamethasone, fluocinolone, a plant
alkaloid, a hormonal compound and/or antagonist, a biological
response modifier, such as a lymphokine or interferon, an antisense
oligonucleotide or oligonucleotide derivative, shRNA, siRNA, or a
pharmaceutically acceptable salt thereof.
In some embodiments, the candidate therapeutic agent is
acetaminophen, lithium, acyclovir, amphotericin B, an
aminoglycoside, a beta lactam, foscavir, ganciclovir, pentamidine,
a quinolone, a sulfonamide, vancomycin, rifampin, adefovir,
indinavir, didofovir, tenofovir, methotrexate, lansoprazole,
omeprazole, pantopraxole, allopurinol, phenytoin, ifosfamide,
gentamycin, or zoledronate.
In some embodiments, the candidate therapeutic agent is
radiation.
In some embodiments, the viability or functionality of the renal
tissue cells is determined by measuring an indicator of metabolic
activity.
In some embodiments, the indicator of metabolic activity is
resazurin reduction or tetrazolium salt reduction in the renal
tubule mode compared to a control.
In some embodiments, the viability or functionality of the renal
tissue cells is determined by measuring lactate dehydrogenase (LDH)
activity, gamma glutamyl-transferase (GGT) activity, protease
activity, ATP utilization, SGLT2 activity, glucose uptake activity
or RNA expression compared to a control.
In some embodiments, the viability or functionality of the renal
tissue cells is determined by measuring a renal transport molecule
activity in the model compared to a control.
In some embodiments, the transport molecule activity is excretion
and/or uptake of at least one macromolecule.
In some embodiments, the macromolecule is a protein.
In some embodiments, the viability or functionality of the renal
tissue cells is determined by identifying regeneration of the renal
tubular epithelial cells compared to a control.
In some embodiments, the viability or functionality of the renal
tissue cells is determined by measuring the trans-epithelial
electrical resistance or the passive permeability of the renal
tubule model compared to a control.
In some embodiments, the viability or functionality of the renal
tissue cells is determined by measuring changes in vitamin D
production, changes in angiotensin conversion, alterations to ion
exchange, alterations to pH, alterations to acid/base balance,
alterations to renal tubule barrier function, alterations to the
intrarenal renin/angiotensin system (RAS), alterations in
physiology, alterations in pathology, alterations to transport of
molecules, alterations to sodium-glucose cotransporter-2 (SGLT2)
activity, amounts of interstitial fibrotic tissue, or regeneration
of the renal tubule model compared to a control.
In some embodiments, the viability or functionality of the renal
tissue cells is determining by measuring changes in cytoplasmic
proline-rich tyrosine kinase-2 (Pyk2) expression,
thiazide-sensitive cotransporter (TSC) expression, epidermal growth
factor (EGF) expression, transforming growth factor-alpha
(TGF-.alpha.) expression, stem cell factor (SCF) expression,
transforming growth factor-beta (TGF-.beta.) expression, connective
growth tissue factor (CTGF) expression, complement factor B
expression, toll-like receptor 2 (TLR2) expression, toll-like
receptor 4 (TLR4) expression, interleukin-6 (IL-6) expression,
Class II major histocompatibility complex (MHC) expression,
intercellular adhesion moleculare-1 (ICAM-1) expression, monocyte
chemoattractant protein-1 (MCP-1) expression, or plasminogen
activator inhibitor-1 (PAI-1) compared to a control.
In some embodiments, the viability or functionality of the renal
tissue cells is determined by measuring induction of an apoptotic
pathway, changes in cellular or nuclear morphology, changes in the
number or morphology of mitochondria, changes in mitochondrial
function, secretion of chemokines, secretion of cytokines, changes
in the amount or pattern of deposition of extracellular matrix,
deposition of protein crystals or salt crystals, tubular
regeneration, epithelial-mesenchymal transition, inflammation,
ischemia, ischemia/reperfusion, hyperproliferation, alterations in
gene expression, secretion of biomarkers, or epigenetic
modifications.
In some embodiments, the viability or functionality of the renal
tissue cells is measured over time.
In some embodiments, the renal tubule model has been cultured in a
cell culture medium prior to being contacted with the candidate
therapeutic agent.
In some embodiments, the renal tubule model has been cultured for
at least 3 days in the cell culture medium.
In some embodiments, the diseased donor has a congenital
abnormality, diabetes, an immune complex disease, vascular
sclerosis, renal ablation, renal fibrosis, hypertension,
arterionephrosclerosis, lupus nephritis, vascular disease,
inflammation, hemolytic-uremic syndrome, obstructive nephropathy,
dyslipoproteinemia, recurrent dehydration, reflux nephropathy,
radiation nephropathy, atheroembolic renal disease, scleroderma,
sickle cell anemia, retention of lipids, infection, ischemia, a
transport deficiency, crystal deposition, a genetic disorder, a
chronic system disorder, renal cancer,
In some embodiments, further comprising a genetically modified
cell, wherein the phenotype is induced by the genetically modified
cell.
Also provided is a method of predicting the effective dosing
concentration and dosing schedule of a candidate therapeutic agent,
the method comprising contacting varying concentrations or amounts
of the agent with the three-dimensional, engineered, bioprinted,
biological renal tissue model; and measuring the effect of the
agent on the viability or functionality of the renal tissue cells
over time.
In some embodiments, the method further comprises measuring a
recovery of the renal tissue cells over time to determine a minimum
timing between doses that provide efficacy.
The present invention also provides a method of making a renal
tubule disorder model, the method comprising forming a
three-dimensional, engineered, biological renal tubule disorder
model by contacting a first layer of renal tissue with a second
layer of renal tissue, provided that the first layer of renal
tissue comprises renal interstitial tissue, the second layer of
renal tissue comprises epithelial tissue, and at least one renal
tissue layer comprises a bio-ink having diseased cells.
In some embodiments, the diseased cells of the bio-ink comprise
genetically modified cells specific to a disease.
In some embodiments, the genetically modified cells are genetically
modified stem cells.
In some embodiments, the genetically modified cells are genetically
modified fibroblast cells, endothelial cells, epithelial cells, or
any combination thereof. In some embodiments, the genetically
modified cells include a polycystic mutation in a transporter, a
retrovirus, a CRISPR, a viral transduction, a chemical mutagenesis,
or any combination thereof.
In some embodiments, the diseased cells of the bio-ink comprise
cells isolated from a donor with a specific disease.
In some embodiments, the donor has a genetic dysfunction
corresponding to the specific disease.
In some embodiments, the cells isolated from the donor are induced
pluripotent stem cells.
In some embodiments, at least a second renal tissue layer comprises
a bio-ink having diseased cells.
In some embodiments, the renal interstitial tissue comprises renal
fibroblasts and endothelial cells, and the renal epithelial tissue
comprises renal tubular epithelial cells.
The present invention also provides a method of making a renal
tubule disorder model, the method comprising conditioning a
three-dimensional, engineered, bioprinted, biological renal tubule
model to produce a phenotype characteristic of a desired renal
tubule disorder, wherein the renal tubule model comprises: a layer
of renal interstitial tissue, the renal interstitial tissue
comprising renal fibroblasts and endothelial cells; and a layer of
renal epithelial tissue, the renal epithelial tissue comprising
renal tubular epithelial cells, to form the three-dimensional,
engineered, biological renal tubule model; provided that the
interstitial tissue comprises an interstitial bio-ink, the
epithelial tissue comprises an epithelial bio-ink, and form a
three-dimensional, engineered, biological renal tubule model.
In some embodiments, the conditioning step is genetically modifying
the cells to produce the phenotype characteristic of the desired
renal tubule disorder.
In some embodiments, the cells are genetically modified by a
polycystic mutation in a transporter, a retovirus, a CRISPR, a
viral transduction, a chemical mutagenesis, or any combination
thereof.
In some embodiments, the conditioning step includes contacting the
three-dimensional, engineered, biological renal tubule model with
an agent capable of inducing the phenotype characteristic of the
desired renal tubule disorder.
In some embodiments, the agent is a toxicant. In some embodiments,
the toxicant includes one or more of the following: anti-infective,
antibiotics, antibacterials, antifungals, antivirals,
acetaminophen, lithium, acyclovir, amphotericin B, an
aminoglycoside, a beta lactam, foscavir, ganciclovir, pentamidine,
a quinolone, a sulfonamide, vancomycin, rifampin, adefovir,
indinavir, didofovir, tenofovir, methotrexate, lansoprazole,
omeprazole, pantopraxole, allopurinol, phenytoin, ifosfamide,
gentamicin, zoledronate, or any combination thereof.
In some embodiments, the agent is a glucose.
In some embodiments, the agent is a microorganism.
In some embodiments, the microorganism is a bacteria, a virus, a
fungi, a protozoa, or a helminth.
In some embodiments, the agent is an inflammation stimulator.
In some embodiments, the conditioning step is reducing oxygen to
the three-dimensional, engineered, biological renal tubule
model.
In some embodiments, the conditioning step is applying radiation to
the three-dimensional, engineered, biological renal tubule
model.
In some embodiments, the conditioning step is caused by a diabetic
condition.
In some embodiments, the diabetic condition is type 2 diabetes.
In some embodiments, the conditioning step is applying a high
concentration of glucose, high blood pressure, or any combination
thereof.
In some embodiments, the conditioning step is causing a renal
lesion.
In some embodiments, the conditioning step is applying a
carcinogen.
In some embodiments, the conditioning step is causing
inflammation.
In some embodiments, the conditioning step is reducing blood supply
to the three-dimensional, engineered, biological renal tubule
model.
In some embodiments, the conditioning step is applying changes to a
mitochondria.
In some embodiments, the conditioning step is changing cell
morphology.
In some embodiments, the conditioning step is a
hyperproliferation.
In some embodiments, the conditioning step is an epigenetic
modification.
In some embodiments, the conditioning step is depositing
crystals.
In some embodiments, the crystals is one or more of the following:
cholesterol crystals, cholesterol monosodium urate, calcium
oxalate, calcium phosphate hydroxyapatite, 2,8-dihydroxyadenine,
uromodulin, myoglobin-uromodulin, indinavir, acyclovir, a polymyxin
(e.g., polysporin, neosporin, polymyxin B, or polymyxin E),
sulfadiazine, cysteine, uric acid, or magnesium ammonium
phosphate.
In some embodiments, the conditioning step is an accumulation of
proteins, salts, or other precipitous matter.
In some embodiments, the desired renal tubule disorder is an acute
renal disorder.
In some embodiments, the desired renal tubule disorder is a chronic
renal disorder.
In some embodiments, the desired renal tubule disorder is a renal
cancer.
In some embodiments, the acute renal disorder arises from a chronic
renal disorder.
BRIEF DESCRIPTION OF THE DRAWINGS
The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
FIGS. 1A-1B illustrate a 3D model of the PT tubulointerstitial
interface printed with the NovoGen Bioprinter.RTM. instrument. FIG.
1A is a schematic diagram showing a multicellular interstitial
layer underlying a basement membrane that supports an epithelial
monolayer. FIG. 1B is a macroscopic view of 3D PT tissues
positioned on Transwell.RTM. inserts in a 24-well plate.
FIGS. 2A-2F are micrographs showing the histological
characterization of 3D PT tissues. Representative images of tissues
cultured for 14 days are shown. FIG. 2A depicts a micrograph of an
H&E stain showing fully cellular tissue and organization of
interstitial and epithelial layers. FIG. 2B depicts a micrograph of
Gomori's trichrome stain showing deposition of collagen throughout
the tissue. FIG. 2C depicts a micrograph showing the interstitial
layer demonstrating extensive endothelial cell-lined networks
(CD31). FIG. 2D depicts a micrograph showing that renal proximal
tubule epithelial cells (RPTEC) form a monolayer and express
cytokeratin 18. FIG. 2E depicts a micrograph showing that a
collagen IV-rich basement membrane underlies the epithelial cells
and E- cadherin localizes to tight junctions between the cells.
FIG. 2F depicts a micrograph showing that Na+K+ATPase localizes to
the basolateral membrane of RPTEC.
FIGS. 3A-3B are graphs showing the viability and metabolic activity
of 3D PT tissues over time. FIG. 3A is a graph showing the
metabolic activity of 3D tissues assessed by reduction of
alamarBlue.TM.. Data shown represent the relative fluorescence over
time. FIG. 3B is a graph showing GGT function in 3D PT tissues
(blue) or 3D interstitium-only tissues (black). Data shown
represent the standard deviation of the average GGT activity in
mIU/ml calculated from a standard curve. Data shown is the mean of
duplicate measurements from at least 9 independent tissue samples
plus or minus the standard error of the mean.
FIGS. 4A-4B are bar graphs showing the RAS pathway component
activity in 3D PT tissues. FIG. 4A is a bar graph showing
expression levels of ACE in supernatant and lysates from 3D PT
tissues cultured 4 or 14 days. FIG. 4B is a bar graph showing
detection of angiotensin II following ACE-mediated conversion of
exogenous angiotensin I. Data shown is the mean of duplicate
measurements from 3 independent tissue samples plus or minus the
standard error of the mean.
FIGS. 5A-5B are a micrograph and a bar graph, respectively, showing
SGLT2 transporter localization and function. FIG. 5A shows 3D PT
tissues stained with antibodies against SGLT2 after 14 days in
culture. FIG. 5 B shows results when tissues were assessed for
retention of the non-metabolizable glucose analog 2-DG in a
colorimetric assay in the presence or absence of the glucose uptake
inducer insulin or the SGLT2 inhibitor canalgliflozin (Cana).
Starved tissues are indicated Data shown is the mean of triplicate
measurements across 6 independent tissue samples plus or minus the
standard error of the mean. * indicates p<0.05 between the
groups compared by one-way ANOVA.
FIGS. 6A-6E are micrographs and a graph (FIG. 6E) showing P-gp
transporter localization and function. FIG. 6A shows 3D PT tissues
stained with antibodies against P-gp after 14 days in culture.
Tissues were also exposed to 5 pM zosuquidar alone (FIG. 6B), 10 pM
rhodamine 123 (FIG. 6C), or rhodamine 123+zosuquidar for 2 h (FIG.
6D). Tissues were snap fixed, cryosectioned, and all tissues were
imaged at the same exposure time. FIG. 6E depicts a graph showing
fluorescence intensity quantified in FIGS. 6B-6D. Data shown
represents the mean of duplicate measurements from at least 6
independent tissue samples plus or minus the standard error of the
mean. * indicates p<0.05 between the groups as compared by
one-way ANOVA.
FIGS. 7A and 7B are graphs showing that cisplatin decreases overall
viability and epithelial function in 3D PT tissues. Tissues were
treated daily for 7 days with increasing doses of cisplatin. FIG.
7A is a graph showing overall tissue viability measured by
alamarBlue' metabolism. Data shown is indicative of duplicate
measurements from 3 individual tissues. * indicates p<0.05
compared to vehicle control by one-way ANOVA and Dunnett's
post-test. FIG. 7B is a graph showing epithelial viability assessed
by GGT activity. Data shown is the mean of duplicate measurements
from 3 independent tissue samples plus or minus the standard error
of the mean. * indicates p<0.05 compared to vehicle control by
one-way ANOVA and Dunnett's post-test.
FIGS. 8A-8C are graphs showing rescue of cisplatin-induced toxicity
by OCT2 inhibition. 3D PT tissues were dosed daily for 7 days with
vehicle, cimetidine alone, 5 .mu.M cisplatin, or cisplatin with
cimetidine. FIG. 8A depicts alamarBlue' analysis of overall tissue
metabolic activity. FIG. 8B depicts GGT activity as a measure of
epithelial-specific function. FIG. 8C depicts daily LDH release as
a measure of toxicity. For each graph, data shown represents the
mean of duplicate measurements from 3 independent tissue samples
plus or minus the standard error of the mean. * indicates p<0.05
between groups compared as assessed by one-way or two-way ANOVA. In
FIG. 8C, black * indicates p<0.5 compared to between groups
being compared. Colored stars indicate p<0.05 for condition
compared to vehicle.
FIGS. 9A-9D are micrographs showing histological analysis of
cisplatin toxicity. Representative H&E images are shown for
tissues dosed daily for 7 days with vehicle (FIG. 9A), 1 mM
cimetidine (FIG. 9B), 5 .mu.M cisplatin (FIG. 9C), or 5 .mu.M
cisplatin+1 mM cimetidine (FIG. 9D).
FIGS. 10A-10D are micrographs showing the proliferation of RPTEC in
response to damage. Tissues were dosed daily for 7 days with
vehicle (FIG. 10A), 2.5 .mu.M cisplatin (FIG. 10B), 5 .mu.M
cisplatin (FIG. 10C), or 5 .mu.M cisplatin+1 mM cimetidine (FIG.
10D) and stained with an antibody against proliferating cell
nuclear antigen (PCNA). Proliferating cells are marked with white
arrows.
FIGS. 11A-11E are a bar graph (FIG. 11A) and micrographs showing
transporter gene expression and cell morphology for 2D primary
human RPTEC. FIG. 11A depicts relative expression levels of P-gp,
BCRP, megalin, cubilin, SGLT2, OAT1, OAT3, OCT2 and MATE1 for 4
different commercially available sources of primary RPTEC. FIGS.
11B-11E depict representative micrographs of commercially available
primary RPTEC.
FIGS. 12A-12D are micrographs showing the histological
characterization of ExVive.TM. kidney tissues. FIG. 12A depicts the
cell morphology of untreated tissue at 20.times. magnification
after 28 days. FIG. 12B depicts the cell morphology of tissue
treated with 10 g/L glucose after 28 days (14 days cultured and 14
days treated) at 20.times. magnification. FIG. 12C depicts the cell
morphology of untreated tissue at 40.times. magnification after 28
days. FIG. 12D depicts the cell morphology of tissue treated with
10 g/L glucose after 28 days at 40.times. magnification.
Glycogenated nuclei in the epithelial layer are shown by the arrows
in FIGS. 12B and 12D. The insert in FIG. 12D is a magnified view of
the cell indicated by the rightmost arrow in the figure.
FIGS. 13A-13C are micrographs showing the histological
characterization of ExVive.TM. kidney tissues. FIG. 13A depicts the
cell morphology of untreated tissue. FIG. 13B depicts the cell
morphology of tissue treated with a nephrotoxic agent under lower
magnification. FIG. 13C depicts the cell morphology of tissue
treated with a nephrotoxic agent under higher magnification. The
arrows in FIGS. 13B and 13C indicate calcium oxalate deposits in
the tissue.
FIGS. 14A-14B are graphs that depict viability and the epithelial
cell functions in a
Renal Fibrosis-induced ExVive.TM. Human Kidney Tissue that was
treated with TGF.beta.. FIG. 14A depict the viability of the
ExVive.TM. Human Kidney Tissue Treated with TGF.beta.. FIG. 14B
depict the epithelial cell functions in the ExVive.TM. Human Kidney
Tissue Treated with TGF.beta..
FIG. 15 is a bar graph that shows in the ExVive.TM. Human Kidney
Tissue treated with TGF.beta.TGF.beta. induces fibrosis-related
gene expression.
FIGS. 16A-16E are micrographs that show in the ExVive.TM. Human
Kidney Tissue treated with TGF.beta., TGF.beta. induces tissue
thickening and increased extracellular matrix deposition. FIG. 16A
is a micrograph of the control vehicle. FIG. 16B is a micrograph of
ExVive.TM. Human Kidney Tissue treated with 0.37 ng/mL TGF.beta..
FIG. 16C is a micrograph of ExVive.TM. Human Kidney Tissue treated
with 1.1 ng/mL TGF.beta.. FIG. 16D is a micrograph of ExVive.TM.
Human Kidney Tissue treated with 3.3 ng/mL TGF.beta.. FIG. 16E is a
micrograph of ExVive.TM. Human Kidney Tissue treated with 10 ng/mL
TGF.beta..
FIG. 16F is a bar graph that shows in the ExVive.TM. Human Kidney
Tissue treated with TGF.beta., TGF.beta. induces tissue thickening
and increased extracellular matrix deposition.
FIG. 17 is a micrograph that show increased soluble cytokeratin 18
(CK18) following cisplatin treatment with the ExVive.TM. Human
Kidney Tissue.
FIG. 18A-I are graphs that show human renal cortex samples (KT1 and
KT2), ExVive.TM. Human Kidney Tissue (3D-1 and 3D-2), and plated 2D
RPTEC cells (2D RPTEC lot 1105) analyzed for transporter expression
by LC-MS/MS: for P-gp expression (FIG. 18A); for MATE1 expression
(FIG. 18B); for OAT2 expression (FIG. 18C); for OAT1 expression
(FIG. 18D); for MATE2 expression (FIG. 18E); for OAT4 expression
(FIG. 18F); for OAT3 expression (FIG. 18G); for OCT2 expression
(FIG. 18H); and for BCRP expression (FIG. 18I).
DETAILED DESCRIPTION OF THE INVENTION
Certain Definitions
Unless otherwise defined, all technical terms used herein have the
same meaning as commonly understood by one of ordinary skill in the
art to which this invention belongs. As used in this specification
and the appended claims, the singular forms "a," "an," and "the"
include plural references unless the context clearly dictates
otherwise. Any reference to "or" herein is intended to encompass
"and/or" unless otherwise stated.
As used herein, "about" means .+-.10% of the recited value. For
example, about 10 includes 9-11.
As used herein, "array" means a scientific tool including an
association of multiple elements spatially arranged to allow a
plurality of tests to be performed on a sample, one or more tests
to be performed on a plurality of samples, or both. In some
embodiments, a plurality of the renal tubule models are configured
to form an array. In some embodiments, the arrays are adapted for,
or compatible with, screening methods and devices, including those
associated with medium-or high-throughput screening. In further
embodiments, an array allows a plurality of tests to be performed
simultaneously. In further embodiments, an array allows a plurality
of samples to be tested simultaneously. In some embodiments, the
arrays are cellular microarrays. In further embodiments, a cellular
microarray is a laboratory tool that allows for the multiplex
interrogation of living cells on the surface of a solid support. In
other embodiments, the arrays are tissue microarrays. In further
embodiments, tissue microarrays include a plurality of separate
tissues or tissue samples assembled in an array to allow the
performance of multiple biochemical, metabolic, molecular, or
histological analyses (Murphy et al., 2013). In some embodiments,
the array is present in the wells of a microtiter plate.
As used herein, "assay" means a procedure for testing or measuring
the presence or activity of a substance (e.g., a chemical,
molecule, biochemical, protein, hormone, or drug, etc.) in an
organic or biologic sample (e.g., cell aggregate, tissue, organ,
organism, etc.).
As used herein, "basement membrane" means an extracellular matrix
which may comprise collagen IV, laminin-entactin/nidogen complexes,
and proteoglycans (Paulsson, 1992).
As used herein, "biocompatible membrane" means a membrane that is
not toxic to tissue.
As used herein, "bio-ink" means a liquid, semi-solid, or solid
composition for use in bioprinting. In some embodiments, bio-ink
comprises cell solutions, cell aggregates, cell-comprising gels,
multicellular bodies, or tissues. In some embodiments, the bio-ink
additionally comprises non-cellular materials that provide specific
biomechanical properties that enable bioprinting. In some
embodiments, the bio-ink comprises an extrusion compound. In some
cases, the extrusion compound is engineered to be removed after the
bioprinting process. In other embodiments, at least some portion of
the extrusion compound remains entrained with the cells
post-printing and is not removed. An interstitial bio-ink comprises
at least one cell of interstitial origin such as a fibroblast,
mesenchymal cell, or pluripotent cells induced to have interstitial
characteristics. An epithelial bio-ink comprises at least one
epithelial cell type including cells of the proximal tubule.
As used herein, "bioprinting" means utilizing three-dimensional,
precise deposition of cells (e.g., cell solutions, cell-containing
gels, cell suspensions, cell concentrations, multicellular
aggregates, multicellular bodies, etc.) via methodology that is
compatible with an automated or semi-automated, computer-aided,
three-dimensional prototyping device (e.g., a bioprinter). Suitable
bioprinters include the Novogen Bioprinter.RTM. from Organovo, Inc.
(San Diego, Calif.) and those described in U.S. Pat. No. 9,149,952
and U.S. Publ Appl. Nos. 2015/0093932, 2015/0004273, and
2015/0037445.
As used herein, "fibrotic tissue" refers to renal interstitial
tissue that has undergone fibrosis (Farris and Colvin, 2012).
Fibrosis may include both quantitative and qualitative changes to
the renal interstitium and may involve multiple extracellular
components as well as various cell types, including, but not
limited to, fibroblasts, fibrocytes, lymphocytes, monocytes,
macrophages, dendritic cells, mast cells, endothelial cells, and
tubular epithelial cells (Zeisberg and Kalluri, 2015; Farris and
Colvin, 2012).
As used herein, "layer" means an association of cells in X and Y
planes that is one or multiple cells thick. In some embodiments,
the renal tubules describe herein include one layer. In other
embodiments, the renal tubules describe herein include a plurality
of layers. In various embodiments, a layer forms a contiguous,
substantially contiguous, or non-contiguous sheet of cells. In some
embodiments, each layer of renal tubule described herein comprises
multiple cells in the X, Y, and Z axes.
As used herein, "polarized" means spatially asymmetric (Bryant and
Mostov, 2008).
As used herein, "scaffold" refers to synthetic scaffolds such as
polymer scaffolds and porous hydrogels, non-synthetic scaffolds
such as pre-formed extracellular matrix layers, dead cell layers,
and decellularized tissues, and any other type of pre-formed
scaffold that is integral to the physical structure of the
engineered tissue and not able to be removed from the tissue
without damage/destruction of said tissue. In further embodiments,
decellularized tissue scaffolds include decellularized native
tissues or decellularized cellular material generated by cultured
cells in any manner; for example, cell layers that are allowed to
die or are decellularized, leaving behind the extracellular matrix
(ECM) they produced while living. The term "scaffoldless,"
therefore, is intended to imply that pre-formed scaffold is not an
integral part of the engineered tissue at the time of use, either
having been removed or remaining as an inert component of the
engineered tissue. "Scaffoldless" is used interchangeably with
"scaffold-free" and "free of preformed scaffold."
As used herein a "subject" is an organism of any mammalian species
including but not limited to humans, primates, apes, monkey, dogs,
cats, mice, rats, rabbits, pigs, horses and others. A subject can
be any mammalian species alive or dead. Subject includes recently
deceased subjects or biopsy samples taken from a living
subject.
As used herein "therapeutic substance" means any molecule,
biologic, compound or composition that is approved to treat a
disease, under investigation to treat a disease, or that elicits a
biological response such as changes in DNA, RNA, peptide,
polypeptide or protein.
As used herein, "tissue" means an aggregate of cells.
As used herein "viable" means that at least 50% of the cells are
alive. In other embodiments, viable cells are at least 60%, 70%,
80%, 90%, 95%, 97% or more of cells in a bio-ink or tissue layer as
determined by at least one test of viability. Tests for viability
are known in the art, and include the alamarBlue.TM. assay
performed according to the manufacturer's protocol (Thermo Fisher,
Carlsbad, Calif.).
Composition of the Renal Tubule Model
In some embodiments, the cells within the tissues are organized
spatially to recapitulate the laminar architecture of the
tubule-interstitial tissue interface; a polarized tubular
epithelium is present on top of a layer of renal interstitial
tissue that includes an endothelial cell-based microvascular
network. Specialized cells, such as EPO-producing cells, are
optionally included within the peritubular spaces. In some
embodiments, the epithelium possesses or generates brush
borders.
In particular, non-limiting embodiments, the engineered renal
tissues described herein comprise two major parts: 1) an
interstitial layer composed of adult renal fibroblasts and human
umbilical vein endothelial cells (HUVEC); and 2) a polarized
epithelial monolayer composed of either normal human renal proximal
tubule epithelial cells
(RPTEC), Madin-Darby canine kidney cells (MDCK), rat primary RPTEC
cells, and/or immortalized RPTEC cells, wherein immortalization is
optionally achieved through genetic manipulation ofhTERT to form
hTERT-immortalized RPTEC cells. The cells are deposited using the
Organovo NovoGen Bioprinter.RTM. in such a way that the epithelial
layer is apical to the interstitial layer (see FIG. 1A). Structures
are created by spatially-controlled deposition of cells mixed with
a thermo-responsive hydrogel that degrades over time (Novogel.RTM.
2.0) combined with deposition of aerosolized cellular materials by
compressed gas propulsion (inkjet spray). In this embodiment, the
two layers together model the wall of a renal distal tubule. This
configuration is critical for modeling in vivo tissues and
predicting native tissue responses. Response of the epithelial
layer is predictive of native tissue response to drugs, chemicals,
or biological agents, and may provide information relative to
toxicity or efficacy. The interstitial layer is critical for proper
functioning of the epithelium and serves as a model for native
tissue fibrosis, in particular renal tubulointerstitial
fibrosis.
In a particular embodiment, an interstitial layer is bioprinted,
using continuous deposition techniques. In this embodiment, an
epithelial layer is bioprinted, using ink-jet deposition techniques
onto the interstitial layer. A substantially contiguous layer of
epithelium is consistent with in vivo tissues and is critical to
replicate a physiologically relevant architecture. Ink-jet
deposition techniques provide the ability to deposit one or more
thin layers of epithelial cells onto the potentially irregular
surface of the interstitial layer. In such embodiments, ink-jet
deposition of the epithelial layer is optionally performed
immediately after bioprinting of the interstitial layer or after
the interstitial layer has been allowed to mature.
In some embodiments, the cells are bioprinted. In further
embodiments, the bioprinted cells are cohered to form the
engineered renal tubule models. In still further embodiments, the
engineered renal tubule models are free or substantially free of
pre-formed scaffold at the time of fabrication or the time of use.
In some cases, bioprinting allows fabrication of tissues that mimic
the appropriate cellularity of native tissue.
In some embodiments, the three-dimensional, engineered renal tubule
models described herein are distinguished from tissues fabricated
by prior technologies by virtue of the fact that they are
three-dimensional, free of pre-formed scaffolds, consist
essentially of cells, and/or have a high cell density (e.g.,
greater than 30% cellular, greater than 40% cellular, greater than
50% cellular, greater than 60% cellular, greater than 70% cellular,
greater than 80% cellular, greater than 90% cellular, or greater
than 95% cellular).
In some embodiments, the three-dimensional, engineered renal tubule
models described herein are distinguished from native (e.g.,
non-engineered) tissues by virtue of the fact that they are
non-innervated (e.g., substantially free of nervous tissue),
substantially free of mature vasculature, and/or substantially free
of blood components. For example, in various embodiments, the
three-dimensional, engineered renal tubule models are free of
plasma, red blood cells, platelets, and the like and/or
endogenously-generated plasma, red blood cells, platelets, and the
like. In certain embodiments, the engineered renal tubule model
lacks immune cells such as T cell, B cells, macrophages, dendritic
cells, basophils, mast cells or eosinophils. In some embodiments,
the model is not tubular in shape like a naturally occurring renal
proximal tubule, but is planar or sheet-like, this advantageously
allows for in vitro assays and analysis. In some embodiments, the
fibroblasts are not of renal origin. In some embodiments, the
endothelial cells are not of renal origin. In some embodiments, the
epithelial cells are not of human origin. In certain embodiments,
the engineered renal tubule model lacks undifferentiated cells. In
certain embodiments, the engineered renal tubule model lacks
undifferentiated renal cells. In some embodiments, the
three-dimensional, engineered renal tubule models described herein
are distinguished from native renal tubule tissues in that they are
flat or substantially planar. In certain embodiments, the
three-dimensional, engineered renal tubule models described herein
possess functional improvements over native renal tubule tissues;
one example is high viability after a sustained amount of time in
culture up to at least 7, 10 or 27 days in culture. In some
embodiments, the cells used in the renal tubule model are
transformed or immortalized. In some embodiments, the cells used in
the renal tubule model are transgenic and contain protein fusions
with fluorescent proteins, like EGFP, GFP, RFP, YFP, or CFP. In
some embodiments, the cells used in the renal tubule model are
transgenic and contain reporter constructs with fluorescent
proteins; like EGFP, GFP, RFP, YFP, GFP; or luminescent proteins
like firefly or renilla luciferase. In certain embodiments, any of
the cells contain a deletion or insertion of 1, 2, 3, 4, 5, 6, 7,
8, 9, 10 genes or more. In some embodiments, the 3D renal tubule
models are chimeras, wherein at least one cell is form a different
mammalian species than any other cell of the 3D renal tubule model.
In some embodiments, the 3D renal tubule models are chimeras,
wherein at least one cell is form a different human donor than any
other cell of the 3D renal tubule model.
Cellular Inputs
In some embodiments, the engineered tissues, arrays, and methods
described herein include a plurality of cell types. In some
embodiments, the renal tubule models comprise a layer of
interstitial tissue comprising mammalian fibroblasts and mammalian
endothelial cells. In various embodiments, suitable endothelial
cells are derived from human umbilical vein (HUVEC), human primary,
human kidney, or from directed differentiation of induced
pluripotent stem cells (iPS) or human embryonic stem cells (hES).
In some embodiments, the fibroblasts are renal interstitial
fibroblasts. In various embodiments, suitable renal interstitial
fibroblasts are derived from primary cells isolated from human
kidney. In some embodiments, the fibroblasts are dermal or vascular
in origin. In some embodiments, one or more of the cellular
components are derived from a non-human mammal. In some
embodiments, the interstitial tissue comprises tumor cells or
cancer cells. In some embodiments, the layer of interstitial tissue
is substantially a monolayer. In some embodiments, the layer of
interstitial tissue comprises a monolayer over 95% of its surface
area. In some embodiments, the layer of interstitial tissue
comprises a monolayer over 90% of its surface area. In some
embodiments, the layer of interstitial tissue comprises a monolayer
over 80% of its surface area. In some embodiments, the layer of
interstitial tissue is greater than 1 cell thick. In some
embodiments, the layer of interstitial tissue is greater than 2
cells thick. In some embodiments, the layer of interstitial tissue
is greater than 3 cells thick. In some embodiments, the layer of
interstitial tissue is greater than 4 cells thick. In some
embodiments, the layer of interstitial tissue is greater than 5
cells thick. In some embodiments, the layer of interstitial tissue
is greater than 10 cells thick. In some embodiments, the layer of
interstitial tissue is greater than 20 cells thick. In some
embodiments, the layer of interstitial tissue is greater than 50
cells thick. In some embodiments, the layer of interstitial tissue
is greater than 100 cells thick. In some embodiments, the layer of
interstitial tissue is 2-100 cells thick. In some embodiments, the
layer of interstitial tissue is greater than 20 .mu.m thick. In
some embodiments, the layer of interstitial tissue is greater than
30 .mu.m thick. In some embodiments, the layer of interstitial
tissue is greater than 40 .mu.m thick. In some embodiments, the
layer of interstitial tissue is greater than 50 .mu.m thick. In
some embodiments, the layer of interstitial tissue is greater than
100 .mu.m thick. In some embodiments, the layer of interstitial
tissue is greater than 200 .mu.m thick. In some embodiments, the
layer of interstitial tissue is greater than 500 .mu.m thick. In
some embodiments, the layer of interstitial tissue is greater than
600 .mu.m thick. In some embodiments, the layer of interstitial
tissue is greater than 1000 .mu.m thick. In some embodiments, the
layer of interstitial tissue is 20 .mu.m-1000 .mu.m thick. In some
embodiments, the layer of interstitial tissue is less than 20 .mu.m
thick. In some embodiments, the layer of interstitial tissue is
less than 30 .mu.m thick. In some embodiments, the layer of
interstitial tissue is less than 40 .mu.m thick. In some
embodiments, the layer of interstitial tissue is less than 50 .mu.m
thick. In some embodiments, the layer of interstitial tissue is
less than 100 .mu.m thick. In some embodiments, the layer of
interstitial tissue is less than 200 .mu.m thick. In some
embodiments, the layer of interstitial tissue is less than 500
.mu.m thick. In some embodiments, the layer of interstitial tissue
is less than 600 .mu.m thick. In some embodiments, the layer of
interstitial tissue is less than 1000 .mu.m thick.
In some embodiments, the renal tubule models comprise a layer of
epithelial tissue comprising mammalian epithelial cells. In further
embodiments, the epithelial cells are renal tubular epithelial
cells (e.g., proximal tubule epithelial cells). In still further
embodiments, suitable renal tubular epithelial cells are primary
isolates or cells derived from the directed differentiation of stem
cells (induced pluripotent stem cell (iPS)-derived and/or human
embryonic stem cell (hES)-derived). In some embodiments, the renal
tubular epithelial cells are Madin-Darby canine kidney (MDCK)
cells. In some embodiments, the renal tubular epithelial cells are
immortalized human cells. In other embodiments, the renal tubular
epithelial cells are immortalized cells such as hTERT-RPTEC cells,
HK-2 cells, LLC-PK1 cells, or OK cells. In some embodiments, the
epithelial cells are derived from a non-human mammal such as, for
example, rat, mouse, pig, or primate. In some embodiments, the
layer of epithelial tissue consists essentially of renal tubule
epithelial cells. In some embodiments, the layer of epithelial
tissue consists essentially of primary renal tubule epithelial
cells. In some embodiments, the layer of epithelial tissue consists
essentially of renal proximal tubule epithelial cells. In some
embodiments, the layer of epithelial tissue consists essentially of
primary renal proximal tubule epithelial cells. In some
embodiments, the layer of renal epithelial tissue is substantially
a monolayer. In some embodiments, renal tubular epithelial cells
are the only cells present in the layer of renal epithelial tissue.
In some embodiments, the layer of epithelial tissue comprises tumor
cells. In some embodiments, the layer of epithelial tissue
comprises renal cell carcinoma cells. In some embodiments, the
layer of epithelial tissue comprises a monolayer over 95% of its
surface area. In some embodiments, the layer of epithelial tissue
comprises a monolayer over 90% of its surface area. In some
embodiments, the layer of epithelial tissue comprises a monolayer
over 80% of its surface area. In some embodiments, the layer of
epithelial tissue is greater than 1 cell thick. In some
embodiments, the layer of epithelial tissue is greater than 2 cells
thick. In some embodiments, the layer of epithelial tissue is
greater than 3 cells thick. In some embodiments, the layer of
epithelial tissue is greater than 4 cells thick. In some
embodiments, the layer of epithelial tissue is greater than 5 cells
thick. In some embodiments, the layer of epithelial tissue is
greater than 10 cells thick. In some embodiments, the layer of
epithelial tissue is greater than 20 cells thick. In some
embodiments, the layer of epithelial tissue is greater than 50
cells thick. In some embodiments, the layer of epithelial tissue is
greater than 100 cells thick. In some embodiments, the layer of
epithelial tissue is 2-100 cells thick. In some embodiments, the
layer of epithelial tissue is greater than 20 .mu.m thick. In some
embodiments, the layer of epithelial tissue is greater than 30
.mu.m thick. In some embodiments, the layer of epithelial tissue is
greater than 40 .mu.m thick. In some embodiments, the layer of
epithelial tissue is greater than 50 .mu.m thick. In some
embodiments, the layer of epithelial tissue is greater than 100
.mu.m thick. In some embodiments, the layer of epithelial tissue is
greater than 200 .mu.m thick. In some embodiments, the layer of
epithelial tissue is greater than 500 .mu.m thick. In some
embodiments, the layer of interstitial tissue is greater than 600
.mu.m thick. In some embodiments, the layer of epithelial tissue is
greater than 1000 .mu.m thick. In some embodiments, the layer of
epithelial tissue is 20-1000 .mu.m thick. In some embodiments, the
layer of epithelial tissue is less than 1000 .mu.m thick. In some
embodiments, the layer of interstitial tissue is less than 600
.mu.m thick. In some embodiments, the layer of epithelial tissue is
less than 500 .mu.m thick. In some embodiments, the layer of
epithelial tissue is less than 200 .mu.m thick. In some
embodiments, the layer of epithelial tissue is less than 100 .mu.m
thick. In some embodiments, the layer of epithelial tissue is less
than 50 .mu.m thick. In some embodiments, the layer of epithelial
tissue is less than 40 .mu.m thick. In some embodiments, the layer
of epithelial tissue is less than 30 .mu.m thick. In some
embodiments, the layer of epithelial tissue is less than 20 .mu.m
thick.
Optionally, the renal tubule models comprise other cell types
(e.g., EPO-producing cells, immune cells, etc.). In some
embodiments, the immune cells are T cells. In some embodiments, the
immune cells are B cells. In some embodiments, the immune cells are
NK cells. In some embodiments, the immune cells are dendritic
cells. In some embodiments, the immune cells are macrophage
cells.
A wide range of cell ratios are suitable. In some embodiments, the
epithelial layer comprises, consists of, or consists essentially of
proximal tubule epithelial cells. In some embodiments, the
fibroblasts and endothelial cells are the only cells present in the
layer of renal interstitial tissue. In some embodiments, the layer
of renal interstitial tissue further comprises fibrotic tissue. In
some embodiments, the interstitial layer comprises, consists of, or
consists essentially of fibroblasts and endothelial cells in
specific ratios. Suitable proportions of fibroblasts include, by
way of non-limiting examples, about 5, 10, 15, 20, 25, 30, 35, 40,
45, 50, 55, 60, 65, 70, 75, 80, 85, 90, and 95% fibroblasts,
including increments therein. Suitable proportions of endothelial
cells include, by way of non-limiting examples, about 5, 10, 15,
20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, and 95%
endothelial cells, including increments therein. In certain
embodiments, the interstitial layer comprises, consists essentially
of, or consists of a specified ratio of fibroblast to endothelial
cells. In certain embodiments, the ratio of fibroblast to
endothelial cells is at least 5:95, 10:90, 15:85, 20:80, 25:75,
30:70, 35:65, 40:60, 45:65, 50:50, 55:45, 60:40, 65:35, 70:30,
75:25, 80:20, 85:15, 90:10 or 95:5, including increments therein.
In certain embodiments, the ratio of fibroblast to endothelial
cells is 5:95 to 95:5. In certain embodiments, the ratio of
fibroblast to endothelial cells is no more than 5:95, 10:90, 15:85,
20:80, 25:75, 30:70, 35:65, 40:60, 45:65, 50:50, 55:45, 60:40,
65:35, 70:30, 75:25, 80:20, 85:15, 90:10 or 95:5, including
increments therein. In certain embodiments, the ratio of fibroblast
to endothelial cells is about 50:50. In certain embodiments, the
ratio of fibroblast to endothelial cells is from about 60:40 to
about 40:60.
A wide range of cell concentrations are suitable for bio-inks.
Bio-inks are suitably prepared for continuous deposition
bioprinting techniques with concentrations of cells including, by
way of non-limiting examples, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160,
170, 180, 190, 200, 225, 250, 275, 300, or more, million cells per
milliliter of bio-ink. In a particular embodiment, bio-ink prepared
for continuous deposition bioprinting comprises about 100-200
million cells/mL. Bio-inks are suitably prepared for ink-jet
deposition bioprinting techniques with concentrations of cells
including, by way of non-limiting examples, about 0.25, 0.5, 1, 2,
3, 5, 10, 15 or more, million cells per milliliter of bio-ink. In a
particular embodiment, bio-ink prepared for ink-jet deposition
bioprinting comprises about 1-5 million cells/mL. In a particular
embodiment, bio-ink prepared for ink-jet deposition bioprinting
comprises about 1-4 million cells/mL. In a particular embodiment,
bio-ink prepared for ink-jet deposition bioprinting comprises about
1-3 million cells/mL. In a particular embodiment, bio-ink prepared
for ink-jet deposition bioprinting comprises about 1-2 million
cells/mL.
In certain embodiments, the renal interstitial bio-ink comprises
between 50 million and 1 billion cells per milliliter. In certain
embodiments, the renal interstitial bio-ink comprises between 50
million and 900 million cells per milliliter. In certain
embodiments, the renal interstitial bio-ink comprises between 50
million and 800 million cells per milliliter. In certain
embodiments, the renal interstitial bio-ink comprises between 50
million and 700 million cells per milliliter. In certain
embodiments, the renal interstitial bio-ink comprises between 50
million and 600 million cells per milliliter. In certain
embodiments, the renal interstitial bio-ink comprises between 50
million and 500 million cells per milliliter. In certain
embodiments, the renal interstitial bio-ink comprises between 50
million and 400 million cells per milliliter. In certain
embodiments, the renal interstitial bio-ink comprises between 50
million and 300 million cells per milliliter. In certain
embodiments, the renal interstitial bio-ink comprises between 50
million and 200 million cells per milliliter. In certain
embodiments, the renal interstitial bio-ink comprises between 75
million and 600 million cells per milliliter. In certain
embodiments, the renal interstitial bio-ink comprises between 100
million and 600 million cells per milliliter. In certain
embodiments, the renal interstitial bio-ink comprises between 100
million and 500 million cells per milliliter. In certain
embodiments, the renal interstitial bio-ink comprises between 100
million and 400 million cells per milliliter. In certain
embodiments, the renal interstitial bio-ink comprises between 100
million and 300 million cells per milliliter. In certain
embodiments, the renal interstitial bio-ink comprises between 100
million and 200 million cells per milliliter. In certain
embodiments, the renal interstitial bio-ink comprises between 100
million and 150 million cells per milliliter.
In certain embodiments, the renal epithelial bio-ink comprises
between 0.25 million and 5 million cells per milliliter. In certain
embodiments, the renal epithelial bio-ink comprises between 0.25
million and 4 million cells per milliliter. In certain embodiments,
the renal epithelial bio-ink comprises between 0.25 million and 3
million cells per milliliter. In certain embodiments, the renal
epithelial bio-ink comprises between 0.25 million and 2 million
cells per milliliter. In certain embodiments, the renal epithelial
bio-ink comprises between 0.25 million and 1 million cells per
milliliter. In certain embodiments, the renal epithelial bio-ink
comprises between 0.5 million and 5 million cells per milliliter.
In certain embodiments, the renal epithelial bio-ink comprises
between 0.5 million and 4 million cells per milliliter. In certain
embodiments, the renal epithelial bio-ink comprises between 0.5
million and 3 million cells per milliliter. In certain embodiments,
the renal epithelial bio-ink comprises between 0.5 million and 2
million cells per milliliter. In certain embodiments, the renal
epithelial bio-ink comprises between 0.5 million and 1 million
cells per milliliter. In certain embodiments, the renal epithelial
bio-ink comprises between 1 million and 5 million cells per
milliliter. In certain embodiments, the renal epithelial bio-ink
comprises between 1 million and 4 million cells per milliliter. In
certain embodiments, the renal epithelial bio-ink comprises between
1 million and 3 million cells per milliliter. In certain
embodiments, the renal epithelial bio-ink comprises between 1
million and 2 million cells per milliliter.
In certain embodiments, the density of the epithelial bio-ink is
less than the density of the interstitial bio-ink. In certain
embodiments, the ratio of the density of the interstitial bio-ink
to the density of the epithelial bio-ink is about 300:1; about
275:1; about 250:1; about 225:1; about 200:1; about 175:1; about
150:1, about 125:1; about 100:1, about 75:1 or about 50:1. In
certain embodiments, the ratio of the density of the interstitial
bio-ink to the density of the epithelial bio-ink ranges from about
300:1 to about 50:1. In certain embodiments, the ratio of the
density of the interstitial bio-ink to the density of the
epithelial bio-ink ranges from about 250:1 to about 75:1. In
certain embodiments, the ratio of the density of the interstitial
bio-ink to the density of the epithelial bio-ink ranges from about
200:1 to about 75:1. In certain embodiments, the ratio of the
density of the interstitial bio-ink to the density of the
epithelial bio-ink ranges from about 150:1 to about 75:1. In
certain embodiments, the ratio of the density of the interstitial
bio-ink to the density of the epithelial bio-ink ranges from about
125:1 to about 75:1.
In certain embodiments, the bio-ink is a viscous liquid. In certain
embodiments, the bio-ink is a semi-solid. In certain embodiments,
the bio-ink is a solid. In certain embodiments, the viscosity of
the bio-ink is greater than 100 centipoise. In certain embodiments,
the viscosity of the bio-ink is greater than 200 centipoise. In
certain embodiments, the viscosity of the bio-ink is greater than
500 centipoise. In certain embodiments, the viscosity of the
bio-ink is greater than 1,000 centipoise. In certain embodiments,
the viscosity of the bio-ink is greater than 2,000 centipoise. In
certain embodiments, the viscosity of the bio-ink is greater than
5,000 centipoise. In certain embodiments, the viscosity of the
bio-ink is greater than 10,000 centipoise. In certain embodiments,
the viscosity of the bio-ink is greater than 20,000 centipoise. In
certain embodiments, the viscosity of the bio-ink is greater than
50,000 centipoise. In certain embodiments, the viscosity of the
bio-ink is greater than 100,000 centipoise. In certain embodiments,
the viscosity of the bio-ink is less than 100 centipoise. In
certain embodiments, the viscosity of the bio-ink is less than 200
centipoise. In certain embodiments, the viscosity of the bio-ink is
less than 500 centipoise. In certain embodiments, the viscosity of
the bio-ink is less than 1,000 centipoise. In certain embodiments,
the viscosity of the bio-ink is less than 2,000 centipoise. In
certain embodiments, the viscosity of the bio-ink is less than
5,000 centipoise. In certain embodiments, the viscosity of the
bio-ink is less than 10,000 centipoise. In certain embodiments, the
viscosity of the bio-ink is less than 20,000 centipoise. In certain
embodiments, the viscosity of the bio-ink is less than 50,000
centipoise. In certain embodiments, the viscosity of the bio-ink is
less than 100,000 centipoise. In certain embodiments, the viscosity
of the bio-ink is 100-100,000 centipoise.
Architectural Features of the Renal Tubule Model
The renal tubule models of the present disclosure can be
architecturally arranged in many configurations. In certain
embodiments, the epithelial tissue and interstitial tissue layers
are separate architecturally distinct layers that are in direct
contact or separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 .mu.m
or more, including increments therein. In certain embodiments, the
separation is due to the secretion and deposition of extracellular
matrix between the two layers, which for the purposes of this
disclosure is considered contact. In normal physiological tissue
cells and cell layers are polarized to have an apical (lumen
facing) surface and a basolateral surface, which faces other cells
or tissue matrix. For the purposes of the renal tubule models
disclosed herein the basolateral surface refers to a surface that
faces another cell, an extracellular matrix or the surface of a
biocompatible membrane or culture vessel. For the purposes of the
renal tubule models disclosed herein the apical surface refers to a
surface that faces away from the surface of a biocompatible
membrane or culture vessel. In some embodiments, the renal tubular
epithelial cells are polarized. In some embodiments, the layer of
renal interstitial tissue possesses an apical and basolateral
surface.
In some embodiments, the renal tubule model further comprises a
biocompatible membrane. In certain embodiments, the basolateral
surface of the interstitial tissue layer is the surface attached to
a biocompatible membrane or culture vessel; and the apical surface
of the interstitial tissue layer is the surface not attached to a
biocompatible membrane or culture vessel. In certain embodiments,
the epithelial tissue layer is deposited onto and forms a layer on
the apical surface of the interstitial tissue layer, thus forming
two architecturally distinct layers. In certain embodiments, the
epithelial tissue and interstitial tissue layers are in continuous
contact. In certain embodiments, between 99%-100% of the epithelial
tissue layer is in continuous contact with the interstitial tissue
layer. In certain embodiments, between 95%-100% of the epithelial
tissue layer is in continuous contact with the interstitial tissue
layer. In certain embodiments, between 90%-100% of the epithelial
tissue layer is in continuous contact with the interstitial tissue
layer. In certain embodiments, 50-99% of the epithelial tissue
layer is in continuous contact with the interstitial tissue layer.
In certain embodiments, between 80%-100% of the epithelial tissue
layer is in continuous contact with the interstitial tissue layer.
In certain embodiments, between 70%-100% of the epithelial tissue
layer is in continuous contact with the interstitial tissue layer.
In certain embodiments, between 60% -100% of the epithelial tissue
layer is in continuous contact with the interstitial tissue layer.
In certain embodiments, between 50%-100% of the epithelial tissue
layer is in continuous contact with the interstitial tissue layer.
In certain embodiments, less than 99% of the epithelial tissue
layer is in continuous contact with the interstitial tissue layer.
In certain embodiments, less than 98% of the epithelial tissue
layer is in continuous contact with the interstitial tissue layer.
In certain embodiments, less than 97% of the epithelial tissue
layer is in continuous contact with the interstitial tissue layer.
In certain embodiments, less than 95% of the epithelial tissue
layer is in continuous contact with the interstitial tissue layer.
In certain embodiments, less than 90% of the epithelial tissue
layer is in continuous contact with the interstitial tissue layer.
In certain embodiments, less than 80% of the epithelial tissue
layer is in continuous contact with the interstitial tissue layer.
In certain embodiments, the epithelial tissue layer completely
covers the apical surface of the interstitial tissue layer. In
certain embodiments, the epithelial tissue layer covers between
99%-100% of the apical surface of the interstitial tissue layer. In
certain embodiments, the epithelial tissue layer covers between
95%-100% of the apical surface of the interstitial tissue layer. In
certain embodiments, the epithelial tissue layer covers between
90%-100% of the apical surface of the interstitial tissue layer. In
certain embodiments, the epithelial tissue layer covers between
80%-100% of the apical surface of the interstitial tissue layer. In
certain embodiments, the epithelial tissue layer covers between
70%-100% of the apical surface of the interstitial tissue layer. In
certain embodiments, the epithelial tissue layer covers between
60%-100% of the apical surface of the interstitial tissue layer. In
certain embodiments, the epithelial tissue layer covers between
50%-100% of the apical surface of the interstitial tissue layer. In
certain embodiments, the epithelial tissue layer covers less than
99% of the apical surface of the interstitial tissue layer. In
certain embodiments, the epithelial tissue layer covers less than
98% of the apical surface of the interstitial tissue layer. In
certain embodiments, the epithelial tissue layer covers less than
97% of the apical surface of the interstitial tissue layer. In
certain embodiments, the epithelial tissue layer covers less than
95% of the apical surface of the interstitial tissue layer. In
certain embodiments, the epithelial tissue layer covers less than
90% of the apical surface of the interstitial tissue layer. In
certain embodiments, the epithelial tissue layer covers less than
80% of the apical surface of the interstitial tissue layer. In
certain embodiments, the epithelial tissue layer covers less than
70% of the apical surface of the interstitial tissue layer. In
certain embodiments, the epithelial tissue layer covers 50-99% of
the apical surface of the interstitial tissue layer.
Architecture of the Epithelial Tissue Layer
Normally an epithelial tissue cell forms tight junctions with
neighboring cells. The tight junctions are marked by the
transmembrane protein family the cadherins. One of these,
E-cadherin, is especially prominent at tight junctions in renal
tissue, and marks their formation. In certain embodiments, the
epithelial tissue layer consists of cells that form tight
junctions. In certain embodiments, substantially all cells in the
epithelial tissue layer form a tight junction with at least one
neighboring cell. In certain embodiments, between 99%-100% of cells
in the epithelial tissue layer form a tight junction with at least
one other cell. In certain embodiments, between 95%-100% of cells
in the epithelial tissue layer form a tight junction with at least
one other cell. In certain embodiments, between 90%-100% of cells
in the epithelial tissue layer form a tight junction with at least
one other cell. In certain embodiments, between 80%-100% of cells
in the epithelial tissue layer form a tight junction with at least
one other cell. In certain embodiments, between 70%-100% of cells
in the epithelial tissue layer form a tight junction with at least
one other cell. In certain embodiments, between 60%-100% of cells
in the epithelial tissue layer form a tight junction with at least
one other cell. In certain embodiments, between 50%-100% of cells
in the epithelial tissue layer form a tight junction with at least
one other cell. In certain embodiments, 50-99% of cells in the
epithelial tissue layer form a tight junction with at least one
other cell.
Viability and Density of the Cell Layers
An advantage of bioprinting by the methods of this disclosure is
that cells can be printed at high density and high viability. In
certain embodiments, the density of the interstitial cell layer is
greater than 1.times.10.sup.6 cells per mL. In certain embodiments,
the density of the interstitial cell layer is at least
5.times.10.sup.6 cells per mL. In certain embodiments, the density
of the interstitial cell layer is at least 10.times.10.sup.6 cells
per mL. In certain embodiments, the density of the interstitial
cell layer is at least 20.times.10.sup.6 cells per mL. In certain
embodiments, the density of the interstitial cell layer is at least
50.times.10.sup.6 cells per mL. In certain embodiments, the density
of the interstitial cell layer is at least 100.times.10.sup.6 cells
per mL. In certain embodiments, the density of the interstitial
cell layer is at least 200.times.10.sup.6 cells per mL. In certain
embodiments, the density of the interstitial cell layer is at least
500.times.10.sup.6 cells per mL. In certain embodiments, the
density of the interstitial cell layer is between about
100.times.10.sup.6 cells per mL and about 900.times.10.sup.6 cells
per mL. In certain embodiments, the density of the interstitial
cell layer is between about 100.times.10.sup.6 cells per mL and
about 700.times.10.sup.6 cells per mL. In certain embodiments, the
density of the interstitial cell layer is between about
100.times.10.sup.6 cells per mL and about 600.times.10.sup.6 cells
per mL. In certain embodiments, the density of the interstitial
cell layer is between about 100.times.10.sup.6 cells per mL and
about 500.times.10.sup.6 cells per mL. In certain embodiments, the
density of the interstitial cell layer is between about
100.times.10.sup.6 cells per mL and about 300.times.10.sup.6 cells
per mL. In certain embodiments, the density of the interstitial
cell layer is between about 100.times.10.sup.6 cells per mL and
about 200.times.10.sup.6 cells per mL. In certain embodiments, the
layer of renal interstitial tissue or layer of renal epithelial
tissue is between 70%-100% living cells by volume. In certain
embodiments, the viability of the interstitial tissue layer is
greater than 99% living cells by volume. In certain embodiments,
the viability of the interstitial tissue layer is greater than 95%
living cells by volume. In certain embodiments, the viability of
the interstitial tissue layer is greater than 90% living cells by
volume. In certain embodiments, the viability of the interstitial
tissue layer is greater than 80% living cells by volume. In certain
embodiments, the viability of the interstitial tissue layer is
greater than 70% living cells by volume. In certain embodiments,
the viability of the interstitial tissue layer is greater than 60%
living cells by volume. In certain embodiments, the viability of
the interstitial tissue layer is greater than 50% living cells by
volume. In certain embodiments, the viability of the interstitial
tissue layer is 50-99% living cells by volume. In certain
embodiments, this viability is maintained for at least 8, 12, 24,
48, 72, 96, or more hours post printing. In certain embodiments,
this viability is maintained for at least 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 21, or more days post printing. In certain
embodiments, the density of the epithelial cell layer is at least
1.times.10.sup.5 cells per mL. In certain embodiments, the density
of the epithelial cell layer is at least 2.times.10.sup.5cells per
mL. In certain embodiments, the density of the epithelial cell
layer is at least 5.times.10.sup.5cells per mL. In certain
embodiments, the density of the epithelial cell layer is at least
1.times.10.sup.6 cells per mL. In certain embodiments, the density
of the epithelial cell layer is at least 5.times.10.sup.6 cells per
mL. In certain embodiments, the density of the epithelial cell
layer is at least 10.times.10.sup.6 cells per mL. In certain
embodiments, the density of the epithelial cell layer is at least
20.times.10.sup.6 cells per mL. In certain embodiments, the density
of the epithelial cell layer is at least 50.times.10.sup.6 cells
per mL. In certain embodiments, the density of the epithelial cell
layer is at least 100.times.10.sup.6 cells per mL. In certain
embodiments, the density of the epithelial cell layer is at least
200.times.10.sup.6 cells per mL. In certain embodiments, the
density of the epithelial cell layer is at least 500.times.10.sup.6
cells per mL. In certain embodiments, the density of the epithelial
cell layer is less than 1.times.10.sup.5 cells per mL. In certain
embodiments, the density of the epithelial cell layer is less than
2.times.10.sup.5cells per mL. In certain embodiments, the density
of the epithelial cell layer is less than 5.times.10.sup.5cells per
mL. In certain embodiments, the density of the epithelial cell
layer is less than 1.times.10.sup.66 cells per mL. In certain
embodiments, the density of the epithelial cell layer is less than
5.times.10.sup.6 cells per mL. In certain embodiments, the density
of the epithelial cell layer is less than 10.times.10.sup.6 cells
per mL. In certain embodiments, the density of the epithelial cell
layer is 10.times.10.sup.6 cells per mL. In certain embodiments,
the viability of the epithelial tissue layer is greater than 99%
living cells by volume. In certain embodiments, the viability of
the epithelial tissue layer is greater than 95% living cells by
volume. In certain embodiments, the viability of the epithelial
tissue layer is greater than 90% living cells by volume. In certain
embodiments, the viability of the epithelial tissue layer is
greater than 80% living cells by volume. In certain embodiments,
the viability of the epithelial tissue layer is greater than 70%
living cells by volume. In certain embodiments, the viability of
the epithelial tissue layer is greater than 60% living cells by
volume. In certain embodiments, the viability of the epithelial
tissue layer is greater than 50% living cells by volume. In certain
embodiments, the viability of the epithelial tissue layer is 50-99%
living cells by volume. In certain embodiments, this viability is
maintained for at least 8, 12, 24, 48, 72, or 96 hours
post-printing. In certain embodiments, this viability is maintained
for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days
post-printing.
Uniformity of Tissue Architecture
One advantage of bioprinting using the methods of this disclosure
is the high degree of uniformity achieved by the process that is
reflected in the corresponding tissue. In certain embodiments, the
thickness of the renal tubule model is substantially uniform. In
certain embodiments, between 99%-100% of the renal tubule model is
within 10% plus or minus of the overall mean thickness of the renal
tubule model. In certain embodiments, between 95%-100% of the renal
tubule model is within 10% plus or minus of the overall mean
thickness of the renal tubule model. In certain embodiments,
between 90%-100% of the renal tubule model is within 10% plus or
minus of the overall mean thickness of the renal tubule model. In
certain embodiments, between 80%-100% of the renal tubule model is
within 10% plus or minus of the overall mean thickness of the renal
tubule model. In certain embodiments, between 70%-100% of the renal
tubule model is within 10% plus or minus of the overall mean
thickness of the renal tubule model. In certain embodiments,
between 99%-100% of the renal tubule model is within 20% plus or
minus of the overall mean thickness of the renal tubule model. In
certain embodiments, between 95%-100% of the renal tubule model is
within 20% plus or minus of the overall mean thickness of the renal
tubule model. In certain embodiments, between 90%-100% of the renal
tubule model is within 20% plus or minus of the overall mean
thickness of the renal tubule model. In certain embodiments,
between 80%-100% of the renal tubule model is within 20% plus or
minus of the overall mean thickness of the renal tubule model. In
certain embodiments, between 70%-100% of the renal tubule model is
within 20% plus or minus of the overall mean thickness of the renal
tubule model. After treatment with a potential toxic agent, the
renal tubule model may become less uniform.
Non-Cellular Components of Bio-Inks and Cell Layers
Often cells or bio-inks that are bioprinted contain excipients or
extrusion compounds that improve their suitability for bioprinting.
Examples of extrusion compounds include, but are not limited to
gels, hydrogels, peptide hydrogels, amino acid-based gels,
surfactant polyols (e.g., Pluronic F-127 or PF-127),
thermo-responsive polymers, hyaluronates, alginates, extracellular
matrix components (and derivatives thereof), collagens, gelatin,
other biocompatible natural or synthetic polymers, nanofibers, and
self-assembling nanofibers. In some embodiments, the extrusion
compound contains a synthetic polymer. In some embodiments, the
extrusion compound contains a non-synthetic polymer that is not
normally associated with mammalian tissues. In some embodiments,
extrusion compounds are removed after bioprinting by physical,
chemical, or enzymatic means. In some embodiments, the bio-inks of
the present disclosure contain 1% or more extrusion compound by
weight. In some embodiments, the renal tubule models of the present
disclosure contain more than 1% extrusion compound by weight. In
some embodiments, the bio-inks of the present disclosure contain
less than 5% extrusion compound by weight. In some embodiments, the
bio-inks of the present disclosure contain between 0%-2% extrusion
compound by weight. In some embodiments, the bio-inks of the
present disclosure contain less than 1% extrusion compound by
weight. In some embodiments, the renal tubule models of the present
disclosure contain between 0%-5% extrusion compound by weight. In
some embodiments, the renal tubule models of the present disclosure
contain less than 2% extrusion compound by weight. In some
embodiments, the renal tubule models of the present disclosure
contain less than 1% extrusion compound by weight. In some
embodiments, the epithelial bio-ink is free from hydrogel. In some
embodiments, the epithelial bio-ink is free from extrusion
compound. In some embodiments, the epithelial bio-ink is free from
synthetic polymers that are used as excipient or extrusion
compounds. In some embodiments, the renal tubule model is free from
synthetic polymers that are used as excipient or extrusion
compounds. In some embodiments, the epithelial cell layer is free
from synthetic polymers that are used as excipient or extrusion
compounds. In some embodiments, the interstitial cell layer is free
from synthetic polymers that are used as excipient or extrusion
compounds.
Print Surfaces
Provided herein are renal tubule models that are attached to a
biocompatible surface. In certain embodiments, the interstitial
tissue layer is printed onto a biocompatible surface. In certain
embodiments, the biocompatible surface is a membrane with a pore
size of 0.4 .mu.m to 10 .mu.m. In certain embodiments, the
biocompatible surface has a pore size of about 1 .mu.m. In certain
embodiments, the biocompatible surface is coated with a composition
to improve cell adherence or viability. In certain embodiments, the
renal tubule modules are printed into 6-well, 12-well, 24-well,
48-well, 96-well, or 384-well plates. In certain embodiments, the
renal tubule modules are printed into tissue culture plates with
diameters of 60, 100 or 150 mm or more. In certain embodiments, the
renal tubule modules are printed into tissue culture flasks or onto
microfluidic chips. In certain embodiments, the renal tubule models
are printed into/onto Transwell.RTM. inserts.
Process for Production of Renal Tubule Models
This disclosure supports methods and processes for fabricating
renal tubule models. In certain embodiments, the product of a
three-dimensional, engineered, biological renal tubule model is
produced by the process of bioprinting. In certain embodiments, at
least one constituent of the product of a three-dimensional,
engineered, biological renal tubule model is produced by the
process of bioprinting. In certain embodiments, the process of
fabricating a three-dimensional, engineered, biological renal
tubule model, comprises: preparing a renal interstitial bio-ink,
the interstitial bio-ink comprising a plurality of interstitial
cell types, the interstitial cell types comprising renal
fibroblasts and endothelial cells; preparing a renal epithelial
bio-ink, the epithelial bio-ink comprising renal tubular epithelial
cells; depositing the renal interstitial bio-ink and the renal
epithelial bio-ink such that the renal epithelial bio-ink forms a
layer on at least one surface of the layer of renal interstitial
bio-ink; and maturing the deposited bio-ink in a cell culture media
to allow the cells to cohere to form the three-dimensional,
engineered, biological renal tubule model. In certain embodiments,
the renal interstitial tissue bio-ink forms a renal interstitial
tissue layer with an apical and basolateral surface. In certain
embodiments, the renal epithelial bio-ink is deposited in contact
with the apical surface of the renal interstitial tissue layer. In
certain embodiments, the renal epithelial bio-ink consists
essentially of renal tubular epithelial cells. In certain
embodiments, the renal epithelial bio-ink consists essentially of
primary renal tubular epithelial cells. In certain embodiments, the
primary renal tubular epithelial cells are isolated from a subject
with a disease that affects kidney function. In certain
embodiments, the primary renal tubular epithelial cells are
isolated from a subject with polycystic kidney disease. In certain
embodiments, the primary renal tubular epithelial cells are
isolated from a subject with diabetes mellitus type II. In certain
embodiments, the renal epithelial bio-ink comprises renal cell
carcinoma cells. In certain embodiments, the renal epithelial
bio-ink is deposited in a monolayer. In certain embodiments, the
renal interstitial tissue bio-ink is deposited in a monolayer. In
certain embodiments, the layer of renal epithelial tissue is
deposited in continuous contact with the layer of renal
interstitial tissue. In certain embodiments, the renal epithelial
bio-ink forms a layer that covers between 50%-100% of the apical
surface of the layer of renal interstitial tissue. In certain
embodiments, the renal epithelial bio-ink forms a layer that covers
between 70%-100% of the apical surface of the layer of renal
interstitial tissue. In certain embodiments, the renal epithelial
bio-ink forms a layer that covers between 90%400% of the apical
surface of the layer of renal interstitial tissue. In certain
embodiments, the renal epithelial bio-ink forms a layer that covers
50-90% the apical surface of the layer of renal interstitial
tissue. In certain embodiments, at least 50% of renal epithelial
cells of the renal epithelial layer form tight junctions with other
renal epithelial cells. In certain embodiments, at least 70% of
renal epithelial cells of the renal epithelial layer form tight
junctions with other renal epithelial cells. In certain
embodiments, at least 90% of renal epithelial cells of the renal
epithelial layer form tight junctions with other renal epithelial
cells. In certain embodiments, 50-90% of renal epithelial cells of
the renal epithelial layer form tight junctions with other renal
epithelial cells. In certain embodiments, the renal tubule model is
between 50 and 500 .mu.m thick. In certain embodiments, the renal
tubule model is about 100 .mu.m thick. In certain embodiments, the
renal epithelial bio-ink further comprises an extrusion compound.
In certain embodiments, the fibroblasts and endothelial cells are
present in the renal interstitial bio-ink at a ratio of about 95:5
to about 5:95 fibroblasts to endothelial cells. In certain
embodiments, the fibroblasts and endothelial cells are present in
the renal interstitial bio-ink at a ratio of about 75:25 to about
25:75 fibroblasts to endothelial cells. In certain embodiments, the
fibroblasts and endothelial cells are present in the renal
interstitial bio-ink at a ratio of about 60:40 to about 40:60
fibroblasts to endothelial cells. In certain embodiments, the
fibroblasts and endothelial cells are present in the renal
interstitial bio-ink at a ratio of about 50:50 fibroblasts to
endothelial cells. In certain embodiments, the renal interstitial
bio-ink further comprises secretory cells. In certain embodiments,
the renal interstitial bio-ink further comprises immune cells. In
certain embodiments, the renal interstitial bio-ink further
comprises an extrusion compound. In certain embodiments, the renal
interstitial bio-ink comprises glomerular cells. In certain
embodiments, the model is fabricated substantially free of
pre-formed scaffold. In certain embodiments, the renal fibroblasts,
endothelial cells, and renal tubular epithelial cells are mammalian
cells. In certain embodiments, either of the renal interstitial
bio-ink or renal epithelial bio-ink forms a planar layer after
deposition. In certain embodiments, the renal tubule model is of a
uniform thickness. In certain embodiments, the renal interstitial
bio-ink is deposited onto a biocompatible membrane. In certain
embodiments, the renal interstitial bio-ink is deposited onto a
biocompatible membrane with a pore size greater than 0.4 In certain
embodiments, the renal interstitial bio-ink is deposited onto a
biocompatible membrane with a pore size of about 1 um. In certain
embodiments, the three-dimensional, engineered, biological renal
tubule models are deposited to form an array. In certain
embodiments, the three-dimensional, engineered, biological renal
tubule models are deposited to form an array configured to allow
between about 20 .mu.m and about 100 .mu.m of space between each
renal tubule model. In certain embodiments, the renal interstitial
bio-ink is between 30%-100% living cells by volume. In certain
embodiments, the renal interstitial bio-ink is between 70%-100%
living cells by volume. In certain embodiments, the renal
interstitial bio-ink is between 90%-100% living cells by volume. In
certain embodiments, the renal interstitial bio-ink is deposited by
extrusion bioprinting. In certain embodiments, the renal epithelial
bio-ink is deposited by ink-jet bioprinting. In certain
embodiments, the renal interstitial bio-ink is not deposited by
ink-jet bioprinting. In certain embodiments, any layer of the renal
tubule model is viable in in vitro culture in culture after 3 days.
In certain embodiments, any layer of the renal tubule model is
viable in in vitro culture after 10 days.
In certain embodiments, the 3D renal tubule models disclosed herein
are produced by an additive manufacturing process. The additive
manufacturing process for 3D tubule models herein allows customized
fabrication of 3D renal tubule models for in vitro purposes. This
is significant in that the tissues are fabricated due to a user
specified design. In certain embodiments, the 3D renal tubule
models contain only the cells that the user specifies. In certain
embodiments, the 3D renal tubule models contain only the cell types
that the user specifies. In certain embodiments, the 3D renal
tubule models contain only the number of cells or concentration of
cells that the user specifies. In certain embodiments, the 3D renal
tubule models contain cells that have been treated with a small
molecule, therapeutic molecule, or therapeutic substance before or
during fabrication. A therapeutic molecule or substance being any
molecule intended to treat a disease or elicit a biological
response. In certain embodiments, the 3D renal tubule models
contain biocompatible or tissue culture plastics, biocompatible
synthetic polymers, cross linkable gels, reversibly cross-linked
gels and other non-cellular constituents.
Maturation of Renal Tubule Models
In certain embodiments, the renal tubule models of the present
disclosure are matured for a certain amount of time after
bioprinting. In certain embodiments, the models are matured for
1-24 hours before use, for example, at least 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 16, 18, 24 hours or more before use. In certain
embodiments, the models are matured for 1-30 days before use, for
example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 days
or more before use. In some embodiments, shipment or transfer of
the tissues is a use. In certain embodiments, the interstitial
layer of the renal tubule model of the present disclosure is
matured for a certain amount of time after bioprinting before
addition of the epithelial layer. In certain embodiments, the
interstitial layer is matured for 1-24 hours before use, for
example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 16, 18, 24
hours or more before use. In certain embodiments, the interstitial
layer is matured for 1-30 days before use, for example, at least 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30 days or more before use. In
some embodiments, shipment or transfer of the tissues is a use. In
some embodiments, the epithelial layer is bioprinted onto the
interstitial layer within 1-24 hours after bioprinting of the
interstitial layer, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 16, 18, 24 hours after bioprinting of the interstitial layer.
In some embodiments, shipment or transfer of the tissues is a use.
In some embodiments, the epithelial layer is bioprinted onto the
interstitial layer within 1-30 days after bioprinting of the
interstitial layer, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30 days after bioprinting of the interstitial layer.
Uses of the Renal Tubule Models
The renal tubule models described herein can be utilized for
multiple applications. In one embodiment, the tissue barrier can be
utilized for toxicology and ADME applications. In one embodiment,
functional features of the renal tubule models include
establishment of a barrier and demonstrating
permeability/absorption (as evidenced by TEER and Lucifer yellow
permeability). These features allow for permeability kinetics
(Papp) and influx/efflux (ab, ba) studies. In another embodiment,
the investigation of active transport and metabolism via key
transporters and metabolic enzymes respectively can be performed
via well-based assays or through detection of substrates and their
metabolites by mass spectrometry. These same techniques can be used
to assess the mechanism of active transport and metabolism of
various drugs and applied compounds. Transport kinetic, efflux rate
and the permeability coefficient of a test substance could
therefore be utilized for correlation to FDA-recommended reference
drugs. Through barrier function and permeability kinetics, the
renal tubules may be used to predict whether there is active
transport of compounds via renal transporters similar to native
tissue, predict the ability of compounds to disrupt the renal
barrier and/or induce renal inflammation, and/or predict the
efficacy of compounds to modulate inflammation.
In some embodiments, the renal tubule models disclosed herein
comprising immune cells are used in the modeling of inflammation
and inflammatory diseases, as well as the impact of immune
modulation on cancer. In one embodiment, the immune cells are
myeloid or lymphoid cells. In another embodiment, the disease
models are compared side by side to normal tissue models, e.g.,
renal tubule models lacking immune cells, comprising immune cells
but not stimulated to activate the immune cells (quiescent), or
lacking immune cells and stimulated with cytokines to mimic an
immune response. In this embodiment, the renal tubule models are
useful for evaluation of inflammation and immune responses. Tissue
constructs comprising immune cells may also be used to study acute
responses. Renal tubule models comprising immune cells may also be
used to model injury and recovery including acute, subchronic, or
chronic dosing of candidate pharmaceutical compounds or therapies.
In another embodiment, the renal tubule models comprising immune
cells are used to evaluate wound healing and fibrosis. Furthermore,
the tissue constructs comprising immune cells may be used to model
microbial/microbiome interactions. The 3D nature of the renal
tubule models allow for enhanced observation of pathogen
invasiveness and translocation. In one embodiment, the renal tubule
models are treated subsequently with candidate pharmaceutical
agents or treatments to reverse or control the inflammatory
effects. Inflammatory signals that may be detected include the
release of cytokines (e.g. IL-8, TNF-.alpha., IL-4, IL-19, IL-13,
IL-17, and/or IFN-gamma), antimicrobial peptides (e.g. beta
defensin, lysozymes, and/or sIgA), endocrine products such as
somatostatin, activation of inflammatory pathways (e.g. JAK/STAT,
and/or NFkB), evaluation of a barrier disruption in response to
inflammation (histology, TEER, Lucifer yellow, Ussing chamber,
and/or other well-based assays), measuring proliferation,
cytotoxicity, tissue damage, or apoptosis (Caspase 8 or Tunel) or
autophagy or re-epithelialization of wounded area, and expression
of key markers and receptors upregulated in response to
stimulation. For any of the phenotypes described, the renal tubule
models may be used to demonstrate the kinetics and magnitude of
onset as well as recovery from perturbation. For example, one can
dose the renal tubule models with a therapeutic agent and measure
the kinetics of absorption in parallel with the kinetics of onset
of tissue damage, and then remove the test agent and measure the
kinetics of clearance of the molecule in the renal tubule and of
recovery from damage. Analysis of these parameters may enable the
prediction of appropriate dosing levels and dosing schedule for
compounds entering the clinic.
In some embodiments, the renal tubule models are used in a model of
a renal disorder.
In some embodiments, the renal tubule models are used in a model of
a renal disorder, wherein the renal tubule models comprise: (a) a
layer of renal interstitial tissue, the renal interstitial tissue
comprising renal fibroblasts and endothelial cells; and (b) a layer
of renal epithelial tissue, the renal epithelial tissue comprising
renal tubular epithelial cells, to form the three-dimensional,
engineered, biological renal tubule model; provided that the
interstitial tissue comprises an interstitial bio-ink, the
epithelial tissue comprises an epithelial bio-ink, and form a
three-dimensional, engineered, biological renal tubule model,
wherein the model manifests a phenotype characteristic of a
disorder associated with the renal tubule.
In some embodiments, the renal tubule models may be used in a
method of assessing the ability of a therapeutic agent to reverse,
reduce, induce, or prevent a renal disorder.
Also provided is a method of assessing the ability of a candidate
therapeutic agent to reverse, reduce, induce, or prevent a renal
disorder, the method comprising: (a) contacting the renal tubule
model with the candidate therapeutic agent; (b) determining the
viability or functionality of the renal tissue cells; and (c)
assessing the ability of the candidate therapeutic to reverse,
reduce, induce, or prevent a renal disease based on the determined
viability or functionality of the renal tissue cells compared to a
control renal tubule model that has not been contacted with the
candidate therapeutic agent.
In some embodiments, the renal tubules and arrays disclosed herein
are for use in in vitro assays. In some embodiments, an "assay" is
a procedure for testing or measuring the presence or activity of a
substance (e.g., a chemical, molecule, biochemical, drug, etc.) in
an organic or biologic sample (e.g., cell aggregate, tissue, organ,
organism, etc.). In further embodiments, assays include qualitative
assays and quantitative assays. In still further embodiments, a
quantitative assay measures the amount of a substance such as a
chemical or biomolecule in a sample.
In various embodiments, the renal tubules and arrays are for use
in, by way of non-limiting example, image-based assays, measurement
of secreted proteins, expression of markers, and production of
proteins or mRNAs. In various further embodiments, the renal
tubules and arrays are for use in assays to detect or measure one
or more of: molecular binding (including radioligand binding),
molecular uptake, activity (e.g., enzymatic activity and receptor
activity, etc.), gene expression, protein expression, protein
modifications (non-limiting examples include: phosphorylation,
ubiquitination, acetylation, glycosylation, lipidation, etc.),
receptor agonism, receptor antagonism, cell signaling, apoptosis,
chemosensitivity, transfection, cell migration, chemotaxis, cell
viability, cell proliferation, safety, efficacy, metabolism,
toxicity, infectivity, and abuse liability. In various embodiments,
the renal tubules are for toxicology, pharmaceutical or toxicity
testing.
In some embodiments, the renal tubules and arrays are for use in
immunoassays.
Immunoassays include, for example, flow cytometry, high throughput
or low throughput image analysis, immunoprecipitation,
radio-immunoassay (RIA), enzyme-linked immunosorbent assays
(ELISA), western blot, homogenous assays, such as A1phaLISA.TM. and
related technologies that rely on time resolved fluorescence or
fluorescence resonance energy transfer (FRET). In further
embodiments, immunoassays are competitive immunoassays or
noncompetitive immunoassays. In a competitive immunoassay, for
example, the antigen in a sample competes with labeled antigen to
bind with antibodies and the amount of labeled antigen bound to the
antibody site is then measured. In a noncompetitive immunoassay
(also referred to as a "sandwich assay"), for example, antigen in a
sample is bound to an antibody site; subsequently, labeled antibody
is bound to the antigen and the amount of labeled antibody on the
site is then measured.
In some embodiments, the renal tubules and arrays are for use in
ELISA. In further embodiments, an ELISA is a biochemical technique
used to detect the presence of an antibody or an antigen in a
sample. In ELISA, for example, at least one antibody with
specificity for a particular antigen is utilized. By way of further
example, a sample with an unknown amount of antigen is immobilized
on a solid support (e.g., a polystyrene microtiter plate) either
non-specifically (via adsorption to the surface) or specifically
(via capture by another antibody specific to the same antigen, in a
"sandwich" ELISA). By way of still further example, after the
antigen is immobilized, the detection antibody is added, forming a
complex with the antigen. The detection antibody is, for example,
covalently linked to an enzyme, or is itself detected by a
secondary antibody that is linked to an enzyme through
bioconjugation.
For example, in some embodiments, an array, microarray, or chip of
cells, multicellular aggregates, or tissues is used for drug
screening or drug discovery. In further embodiments, an array,
microarray, or chip of tissues is used as part of a kit for drug
screening or drug discovery. In some embodiments, each renal tubule
exists within a well of a biocompatible multi-well container,
wherein the container is compatible with one or more automated drug
screening procedures and/or devices. In further embodiments,
automated drug screening procedures and/or devices include any
suitable procedure or device that is computer or
robot-assisted.
In further embodiments, arrays for drug screening assays or drug
discovery assays are used to research or develop drugs potentially
useful in any therapeutic area. In still further embodiments,
suitable therapeutic areas include, by way of non-limiting
examples, infectious disease, hematology, oncology, pediatrics,
cardiology, central nervous system disease, neurology,
gastroenterology, hepatology, urology, infertility, ophthalmology,
nephrology, orthopedics, pain control, psychiatry, pulmonology,
vaccines, wound healing, physiology, pharmacology, dermatology,
gene therapy, toxicology, toxicity, and immunology.
In some embodiments, the renal tubules and arrays are for use in
cell-based screening. In further embodiments, the cell-based
screening is for one or more infectious diseases such as viral,
fungal, bacterial or parasitic infection. In further embodiments,
the cell-based screening is for kidney cancer, including renal cell
carcinoma, juxtaglomerular cell tumor (reninoma), angiomyolipoma,
renal oncocytoma, Bellini duct carcinoma, clear-cell sarcoma of the
kidney, mesoblastic nephroma, Wilms' tumor, mixed epithelial
stromal tumor, and transitional cell carcinoma of the renal pelvis.
In further embodiments, the cell-based screening is for nephritis,
including, glomerulonephritis, interstitial nephritis or
tubulo-interstitial nephritis, pyelonephritis, lupus nephritis and
athletic nephritis. In further embodiments, the cell-based
screening is for hypertension. In further embodiments, the
cell-based screening is for diabetes mellitus, type I, type II and
MODY. In further embodiments, the cell-based screening is for a
nephropathy, including IgA nephropathy, analgesic nephropathy, or
onconephropathy. In some embodiments, the cell-based screening is
for polycystic kidney disease or Xanthine oxidase deficiency. In
other embodiments, the renal tubules and arrays are for use in the
study of cancer initiation, progression, or metastasis. In still
further embodiments, the renal tubules and arrays are for use in
the study of the interaction of other cell types, such as cancer
cells, pathogen-bearing cells, pathogenic cells, immune cells,
blood-derived cells, or stem/progenitor cells.
In some embodiments, the constructs or arrays thereof are for use
in assessing the performance of biologics, including antibodies,
mammalian cells, bacteria, biologically-active proteins, hormones,
etc. In other embodiments, the renal tubules or arrays thereof are
useful in the study of cell-cell and cell-tissue interactions
between the mammalian renal tubules comprising the construct and
one or more additional cell types, including but not limited to
pathogen-bearing cells, living pathogenic cells, cancer cells,
immune cells, blood cells, stem/progenitor cells, or
genetically-manipulated cells.
In some embodiments, the array comprises renal tubules and
additional tissue constructs. In further embodiments, the renal
tubule construct is in direct contact with an additional tissue
construct on one or more surfaces. In still further embodiments,
the renal tubule is connected to one or more additional tissues
constructs or cells via a fluid path or common fluid reservoir. In
still further embodiments, the liquid media that contacts the
engineered renal tubule construct contains living mammalian cells
such as immune cells, blood-derived cells, or tumor-derived cells.
In other embodiments, the liquid media that contacts the renal
tubule contains bacteria, fungi, viruses, parasites, or other
pathogens.
Provided are methods of assessing the ability of a candidate
therapeutic agent to reverse, reduce or prevent renal injury by a
potential toxic agent comprising: contacting the potential toxic
agent with a three-dimensional, engineered, bioprinted, biological
renal tubule model; contacting the renal tubule model with the
candidate therapeutic agent; determining the viability or
functionality of the renal tubular epithelial cells; and assessing
the ability of the candidate therapeutic agent to reverse, reduce
or prevent renal injury by the potential toxic agent based on the
determined viability or functionality of the renal tubular
epithelial cells compared to a control renal tubule model that has
not been contacted with the candidate therapeutic agent. In certain
embodiments, the three-dimensional, engineered, bioprinted,
biological renal tubule model comprises a layer of renal
interstitial tissue and a layer of renal epithelial tissue. In
other embodiments, the renal interstitial tissue comprises renal
fibroblasts and endothelial cells, and the renal epithelial tissue
comprises renal tubular epithelial cells to form the
three-dimensional, engineered, biological renal tubule model;
provided that the interstitial tissue comprises an interstitial
bio-ink, the epithelial tissue comprises an epithelial bio-ink, and
form a three-dimensional, engineered, biological renal tubule
model.
In one embodiment, the fibroblasts and endothelial cells are
present in a ratio of fibroblasts to endothelial cells at which the
renal tubule model is planar six days post-printing. In some
embodiments, the fibroblasts and endothelial cells are present in
the layer of renal interstitial tissue at a ratio of about 50:50
fibroblasts to endothelial cells.
In some embodiments, the model further comprises a layer of
basement membrane between the renal interstitial tissue layer and
the renal epithelial tissue layer. In some embodiments, the layer
of renal epithelial tissue is in continuous contact with the layer
of basement membrane, and the layer of basement membrane is in
continuous contact with the layer of renal interstitial tissue.
In some embodiments, the renal tubular model is at least 3 cell
layers thick. In some embodiments, the renal tubular model is 2 or
more cell layers thick. In some embodiments, the mean thickness of
the renal tubule model is at least 50 .mu.m. In some embodiments,
the mean thickness of the renal tubule model is at least 100 .mu.m.
In some embodiments, the mean thickness of the renal tubule model
is at least 200 .mu.m. In some embodiments, the mean thickness of
the renal tubule model is at least 300 .mu.m. In some embodiments,
the mean thickness of the renal tubule model is at least 400 .mu.m.
In some embodiments, the mean thickness of the renal tubule model
is at least 500 .mu.m. In some embodiments, the mean thickness of
the renal tubule model is at least 600 .mu.m. In some embodiments,
the mean thickness of the renal tubule model is at least 700 .mu.m.
In some embodiments, the mean thickness of the renal tubule model
is at least 800 .mu.m. In some embodiments, the mean thickness of
the renal tubule model is at least 900 .mu.m. In some embodiments,
the mean thickness of the renal tubule model is at least 1000
.mu.m. In some embodiments, the mean thickness of the renal tubule
model is between 50 .mu.m and 1000 .mu.m. In some embodiments, the
mean thickness of the renal tubule model is between 75 .mu.m and
1000 .mu.m. In some embodiments, the mean thickness of the renal
tubule model is between 100 .mu.m and 1000 .mu.m. In some
embodiments, the mean thickness of the renal tubule model is
between 200 .mu.m and 1000 .mu.m. In some embodiments, the mean
thickness of the renal tubule model is between 500 .mu.m and 1000
.mu.m. In some embodiments, the mean thickness of the renal tubule
model is between 50 .mu.m and 500 .mu.m. In some embodiments, the
mean thickness of the renal tubule model is between 50 .mu.m and
300 .mu.m. In some embodiments, the mean thickness of the renal
tubule model is between 50 .mu.m and 200 .mu.m. In some
embodiments, the mean thickness of the renal tubule model is
between 50 .mu.m and 150 .mu.m. In some embodiments, the mean
thickness of the renal tubule model is between 50 .mu.m and 125
.mu.m. In some embodiments, the mean thickness of the renal tubule
model is between 75 .mu.m and 100 .mu.m.
In some embodiments, the surface area of the renal tubule model is
between 0.01 cm.sup.2 and 0.1 cm.sup.2. In some embodiments, the
surface area of the renal tubule model is at least 0.01 cm.sup.2.
In some embodiments, the surface area of the renal tubule model is
at least 0.02 cm.sup.2. In some embodiments, the surface area of
the renal tubule model is at least 0.03 cm.sup.2. In some
embodiments, the surface area of the renal tubule model is at least
0.04 cm.sup.2. In some embodiments, the surface area of the renal
tubule model is at least 0.05 cm.sup.2. In some embodiments, the
surface area of the renal tubule model is at least 0.06 cm.sup.2.
In some embodiments, the surface area of the renal tubule model is
at least 0.07 cm.sup.2. In some embodiments, the surface area of
the renal tubule model is at least 0.08 cm.sup.2. In some
embodiments, the surface area of the renal tubule model is at least
0.09 cm.sup.2. In some embodiments, the surface area of the renal
tubule model is at least 0.10 cm.sup.2. In some embodiments, the
surface area of the renal tubule model is at least 0.11 cm.sup.2.
In some embodiments, the surface area of the renal tubule model is
at least 0.12 cm.sup.2. In some embodiments, the surface area of
the renal tubule model is less than 0.5 cm.sup.2. In some
embodiments, the surface area of the renal tubule model is less
than 0.4 cm.sup.2. In some embodiments, the surface area of the
renal tubule model is less than 0.3 cm.sup.2. In some embodiments,
the surface area of the renal tubule model is less than 0.2
cm.sup.2. In some embodiments, the surface area of the renal tubule
model is less than 0.1 cm.sup.2.
The potential toxic agent is anything that may have an affect on
the structure or function of renal tissue. In some embodiments, the
potential toxic agent is a toxin, a therapeutic agent, an
antimicrobial agent, a metal, or an environmental agent. In other
embodiments, the potential toxic agent is an antiviral, an
analgesic agent, an antidepressant agent, a diuretic agent, or a
proton pump inhibitor.
In other embodiments, the potential toxic agent is a cytokine, a
chemokine, a small molecule drug, a large molecule drug, a protein
or a peptide.
In other embodiments, the potential toxic agent is a
chemotherapeutic agent which is an aromatase inhibitor; an
anti-estrogen; an anti-androgen; a gonadorelin agonist; a
topoisomerase I inhibitor; a topoisomerase II inhibitor; a
microtubule active agent; an alkylating agent; a retinoid, a
carontenoid, or a tocopherol; a cyclooxygenase inhibitor; an MMP
inhibitor; an mTOR inhibitor; an antimetabolite; a platin compound;
a methionine aminopeptidase inhibitor; a bisphosphonate; an
antiproliferative antibody; a heparanase inhibitor; an inhibitor of
Ras oncogenic isoforms; a telomerase inhibitor; a proteasome
inhibitor; a compound used in the treatment of hematologic
malignancies; a Flt-3 inhibitor; an Hsp90 inhibitor; a kinesin
spindle protein inhibitor; a MEK inhibitor; an antitumor
antibiotic; a nitrosourea; a compound targeting/decreasing protein
or lipid kinase activity, a compound targeting/decreasing protein
or lipid phosphatase activity, or an anti-angiogenic compound. In
other embodiments, the potential toxic agent is a chemotherapeutic
agent which is daunorubicin, adriamycin, Ara-C, VP-16, teniposide,
mitoxantrone, idarubicin, cisplatin, carboplatinum, PKC412,
6-mercaptopurine (6-MP), fludarabine phosphate, octreotide, SOM230,
FTY720, 6-thioguanine, cladribine, 6-mercaptopurine, pentostatin,
hydroxyurea, 2-hydroxy-1H-isoindole-1,3-dione derivatives,
1-(4-chloroanilino)-4-(4-pyridylmethyl)phthalazine,
1-(4-chloroanilino)-4-(4-pyridylmethyl)phthalazine succinate,
angiostatin, endostatin, anthranilic acid amides, ZD4190, ZD6474,
SU5416, SU6668, bevacizumab, rhuMAb, rhuFab, macugon, FLT-4
inhibitors, FLT-3 inhibitors, VEGFR-2 IgGI antibody, RPI 4610,
bevacizumab, porfimer sodium, anecortave, triamcinolone,
hydrocortisone, 11-.alpha.-epihydrocotisone, cortex olone,
17a-hydroxyprogesterone, corticosterone, desoxycorticosterone,
testosterone, estrone, dexamethasone, fluocinolone, a plant
alkaloid, a hormonal compound and/or antagonist, a biological
response modifier, such as a lymphokine or interferon, an antisense
oligonucleotide or oligonucleotide derivative, shRNA, siRNA, or a
pharmaceutically acceptable salt thereof.
In other embodiments, the potential toxic agent is acetaminophen,
lithium, acyclovir, amphotericin B, and aminoglycoside, a beta
lactams, foscavir, ganciclovir, pentamidine, a quinolone, a
sulfonamide, vancomycin, rifampin, adefovir, indinavir, didofovir,
tenofovir, methotrexate, lansoprazole, omeprazole, pantopraxole,
allopurinol, phenytoin, ifosfamide, gentamycin, or zoledronate.
In some embodiments, the potential toxic agent is radiation. In
some embodiments, radiation may include X-rays, gamma rays, UV, and
others. In some embodiments, radiation is used alone or in
combination with another toxic agent or agents. In some
embodiments, the radiation may include photon radiotherapy,
particle beam radiation therapy, other types of radiotherapies, and
combinations thereof.
In some embodiments, the toxic agent is dissolved in a
biocompatible solvent. When the potential toxic agent is water
insoluble, the potential toxic agent may be dissolved with a polar,
aprotic organic solvent such as dimethyl sulfoxide (DMSO) or
dimethyl formamide (DMF) and then diluted with a aqueous solution
such as 9 g/L sodium chloride (saline), in distilled water, aqueous
Tween, culture media, or another biocompatible solvent.
In some embodiments, the viability or functionality of the renal
tubular epithelial cells is determined by measuring an indicator of
metabolic activity. In some embodiments, metabolic activity may be
measured by alamarBlue.TM. assay (Thermo Fisher, Carslbad, CA),
lactate dehydrogenase (LDH) activity assay, or another assay. In
some embodiments, the indicator of metabolic activity is resazurin
reduction or tetrazolium salt reduction in the renal tubule mode
compared to a control. In some embodiments, resazurin reduction is
measured using the alamarBlue.TM. assay (Rampersad, 2012). In some
embodiments, the tetrazolium salts include
3-(4,5-dimethyl)thiazol-2-yl)-2, 5-diphenyltetrazolium bromide
(1vITT); sodium 3'-[1-phenylamino)-carbonyl]-3,4-tetrazolium]-bis
(4-methoxy-6-nitrobenzene) sulfonic acid hydrate (XTT);
4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene
disulfonate, water-soluble tetrazolium salt (WST-1); and others
(Rampersad, 2012).
In some embodiments, the viability or functionality of the renal
tubular epithelial cells is determined by measuring lactate
dehydrogenase (LDH) activity (see Example 2), gamma
glutamyl-transferase (GGT) activity (see Example 2), protease
activity, ATP utilization, glucose uptake activity (see Example 7),
sodium-glucose co-transporter-2 (SGLT2) activity (see Example 12),
or RNA expression (see Example 6) compared to a control. In some
embodiments, protease activity is measured by measuring caspase
activity using synthetic peptide substrates (Kumar, 2004). In some
embodiments, intracellular ATP is measured using an ATP assay kit
(Weng, 2015).
In other embodiments, the viability or functionality of the renal
tubular epithelial cells is determined by measuring a renal
transport molecule activity in the model compared to a control. In
other embodiments, the transport molecule activity is excretion
and/or uptake of at least one macromolecule. In other embodiments,
the macromolecule is albumin. In some embodiments, albumin uptake
is measured using fluorescence microscopy and cell lysate
fluorescence (Ferrell, 2012).
In other embodiments, the viability or functionality of the renal
tubular epithelial cells is determined by identifying regeneration
of the renal tubular epithelial cells compared to a control. In one
embodiment, regeneration is identified by visually inspecting the
renal tubular epithelial cells and identifying an increase in the
number of viable cells.
In other embodiments, the viability or functionality of the renal
tubular epithelial cells is determined by measuring the
trans-epithelial electrical resistance (see Example 2) or the
passive permeability (see Example 2) of the renal tubule model
compared to a control.
In other embodiments, the viability or functionality of the renal
tubular epithelial cells is determined by measuring changes in
vitamin D production, changes in angiotensin conversion (see
Example 3), alterations to ion exchange, alterations to pH,
alterations to acid/base balance, alterations to renal tubule
barrier function (see Example 10), or alterations to the intrarenal
renin/angiotensin system (RAS) (see Example 11), alterations in
physiology, alterations in pathology (see Example 5), alterations
to transport of molecules (see Example 12), alterations to
sodium-glucose cotransporter-2 (SGLT2) activity (see Example 12),
amounts of interstitial fibrotic tissue, or regeneration of the
renal tubule model compared to a control.
In other embodiments, the viability or functionality of the renal
tubular epithelial cells is determined by measuring amounts of
interstitial fibrotic tissue compared to a control. In some
embodiments, interstitial fibrotic tissue is measured using
trichrome-PAS fibrosis measurement, collagen III
immunohistochemistry, Sirius Red staining, or another type of assay
(Farris et al., 2011). In some embodiments, the viability or
functionality of the renal tubular epithelial cells is measured
over time.
In some embodiments, the renal tubule model is contacted first with
the potential toxic agent and then with the candidate therapeutic
agent. In other embodiments, the renal tubule model is contacted
first with the candidate therapeutic agent and then with the
potential toxic agent. In some embodiments, the renal tubule model
has been cultured in a cell culture medium prior to being contacted
with the candidate therapeutic agent and the potential toxic agent.
In some embodiments, the renal tubule model has been cultured for
at least 3 days in the cell culture medium.
Also provided are methods of assessing the effect of an agent on
renal function, the method comprising contacting the agent with a
three-dimensional, engineered, bioprinted, biological renal tubule
model and measuring the effect of the agent on renal function the
viability or functionality of the renal tubular epithelial cells.
In some embodiments, the three-dimensional, engineered, bioprinted,
biological renal tubule model comprises a layer of renal
interstitial tissue, the renal interstitial tissue comprising renal
fibroblasts and endothelial cells; and a layer of renal epithelial
tissue, the renal epithelial tissue comprising renal tubular
epithelial cells; provided that the interstitial tissue comprises
an interstitial bio-ink, the epithelial tissue comprises an
epithelial bio-ink, and form a three-dimensional, engineered,
biological renal tubule model. In one embodiment, the fibroblasts
and endothelial cells are present in a ratio of fibroblasts to
endothelial cells at which the renal tubule model is planar six
days post-printing.
Models of Renal Disorders
Provided are models of a renal disorder, comprising a
three-dimensional, engineered, bioprinted, biological renal tubule
model. In some embodiments, the three-dimensional, engineered,
bioprinted, biological renal tubule models comprise a layer of
renal interstitial tissue, the renal interstitial tissue comprising
renal fibroblasts and/or endothelial cells; and a layer of renal
epithelial tissue, the renal epithelial tissue comprising renal
tubular epithelial cells; provided that the interstitial tissue
comprises an interstitial bio-ink, the epithelial tissue comprises
an epithelial bio-ink, and form a three-dimensional, engineered,
biological renal tubule model.
In some embodiments, the renal disorder is associated with
retention of lipids within the renal model. Lipid accumulation can
be induced in the model by incorporation of adipocytes. In some
embodiments, the renal disorder is associated with congenital
abnormality, diabetes, an immune complex disease, vascular
sclerosis, renal fibrosis, hypertension, arterionephrosclerosis,
lupus nephritis, vascular disease, inflammation, hemolytic-uremic
syndrome, obstructive nephropathy, dyslipoproteinemia, recurrent
dehydration, reflux nephropathy, radiation nephropathy,
atheroembolic renal disease, scleroderma, sickle cell anemia,
retention of lipids, toxicant exposure, infection, ischemia,
ischemia/reperfusion, a transport deficiency, a cystic disease, a
crystallopathy, or a combination thereof. In some embodiments, the
renal disorder may arise following an environmental exposure. In
other embodiments, the renal disorder may arise as a result of a
genetic or epigenetic modification. In some embodiments, the renal
disorder may arise following a defect in cellular localization or
activity of a transporter, enzyme, or other protein.
Acute Renal Disorders
In some embodiments, the renal disorder is an acute renal
disorder.
In some embodiments, the acute renal disorder is acute tubular
necrosis. Acute tubular necrosis involves the death of tubular
epithelial cells that form the renal tubules of the kidneys. Acute
tubular necrosis is a form of acute kidney injury that may be
life-threatening.
In some embodiments, the acute renal disorder is acute interstitial
nephritis.
Acute interstitial nephritis is a renal lesion that causes a
decline in renal function and is characterized by the infiltration
and localization of inflammatory cells in the kidney interstitium.
Acute interstitial nephritis is a form of acute kidney injury.
In some embodiments, the acute renal disorder is an acute kidney
injury. Acute kidney injury is also called acute renal failure or
acute kidney failure. Acute kidney injury is an abrupt or rapid
decline in renal filtration. Acute kidney injury occurs when the
kidneys suddenly become unable to filter waste products from the
blood and can result in the accumulation of dangerous levels of
waste.
In some embodiments, the acute kidney injury is caused by toxicant
exposure, diabetes, infection, inflammation, ischemia, or
ischemia/reperfusion.
In some embodiments, the acute kidney injury is caused by toxicant
exposure. In some embodiments, the toxicant is an anti-infective.
Anti-infectives include antibiotics, antibacterials, antifungals,
and antivirals. In some embodiments, the toxicant is an antibiotic,
an antibacterial, an antifungal, or an antiviral. In some
embodiments, the toxicant is acetaminophen, lithium, acyclovir,
amphotericin B, an aminoglycoside, a beta lactam, foscavir,
ganciclovir, pentamidine, a quinolone, a sulfonamide, vancomycin,
rifampin, adefovir, indinavir, didofovir, tenofovir, methotrexate,
lansoprazole, omeprazole, pantopraxole, allopurinol, phenytoin,
ifosfamide, gentamicin, or zoledronate.
In some embodiments, the acute kidney injury is caused by diabetes.
In some embodiments, the diabetes is type 2 diabetes. In some
embodiments, the diabetes is type 1 diabetes. In some embodiments,
the diabetes is caused by exposure to high blood glucose and/or
high blood pressure. In some embodiments, the proximal tubule
injury resulting from diabetes is secondary to a decline in
glomerular integrity and function, which leads to elevated glucose,
proteins such as albumin, or other blood components in the
filtrate. In some embodiments, the kidney injury resulting from
diabetes is caused in part by exposure of the tubule to elevated
levels of glucose, protein, and other blood components in the
filtrate. In some embodiments, the acute kidney injury is caused by
diabetic ketoacidosis. Diabetic ketoacidosis (DKA) is a
life-threatening complication of diabetes (usually type 1 diabetes)
characterized by hyperglycemia, hyperketonemia, and metabolic
acidosis. DKA is usually triggered by insulin deficiency and
hyperglycemia combined with significant physiologic stress, such as
acute infection, myocardial infarction, stroke, pancreatitis, or
trauma. It can also be triggered by corticosteroids, thiazide
diuretics, and sympathomimetics. In some embodiments, tubular
injury is further compromised by loss or compromise of
microvasculature and resulting hypoxia.
In some embodiments, the acute kidney injury is caused by
infection. In some embodiments, the infection is caused by a
microorganism or microbe. In some embodiments, the microorganism
that causes infection is a bacteria, a virus, a fungi, a protozoa,
or a helminth.
In some embodiments, the acute kidney injury is caused by
inflammation. In some embodiments, the inflammation is caused by a
pattern recognition receptor. Pattern recognition receptors include
toll-like receptors (TLRs), retinoic acid-inducible gene
(RIG)-I-like receptors, NOD-like receptors, and C-type lectin
receptors. In some embodiments, the inflammation is caused by a
TLR, a RIG-I-like receptor, a NOD-like receptor, or a C-type lectin
receptor.
In some embodiments, the acute kidney injury is caused by ischemia.
Ischemia is an inadequate blood supply to an organ or part of the
body. In some embodiments, the acute kidney injury is caused by
reperfusion. Reperfusion is injury to the kidney caused when blood
supply returns to the kidney after a period of ischemia.
In some embodiments, the acute kidney injury is a secondary
condition to another disorder. In some embodiments, the acute
kidney injury is caused by acute interstitial nephritis, a cystic
disease, a nephropathy, a crystallopathy/nephrolithiasis, an
infectious disease, exposure to a toxicant, renal cancer, or a
potential toxic agent. In some embodiments, the acute kidney injury
is caused by nephritis which arises from lupus, pyelonephritis, or
onconephritis. In some embodiments, the acute kidney injury is
caused by a genetic disorder such as polycystic disease. In some
embodiments, the acute kidney injury is caused by a nephropathy
such as diabetic nephropathy. In some embodiments, the acute kidney
injury is caused by an infectious disease. In some embodiments, the
acute kidney injury is caused by exposure to a toxicant. In some
embodiments, the acute kidney injury is caused by renal cancer. In
some embodiments, the acute kidney injury is caused by a potential
toxic agent.
In some embodiments, the result of acute kidney injury is necrosis,
apoptosis, nephritis, tubular regeneration, compensatory
proliferation, epithelial-mesenchymal transition (EMT),
inflammation, ischemia, reactive oxygen species, changes in the
mitochondria, changes to cell morphology, changes to nuclear
morphology, hyperproliferation, alterations in gene expression,
secretion of biomarkers, or epigenetic modifications.
In some embodiments, the result of acute kidney injury is necrosis.
In some embodiments, acute kidney injury caused by exposure to
toxicants results in necrosis.
In some embodiments, the result of acute kidney injury is
apoptosis. Apoptosis is the death of cells that occurs as a normal
and controlled part of an organism's growth or development. In some
embodiments, acute kidney injury is caused by exposure to toxicants
resulting in apoptosis.
In some embodiments, the result of acute kidney injury is tubular
regeneration. During tubular regeneration, renal epithelial cells
undergo morphological changes, migrate, and proliferate to replace
lost cells, finally resulting in physiological and functional
recovery of the renal epithelium. Molecules such as vimentin,
Pax-2, and neural cell adhesion molecule can be re-expressed in
renal epithelial cells during recovery from acute kidney injury
(Tang et al., 2015).
In some embodiment, the result of acute kidney injury is
compensatory proliferation of existing tubular cells, which
proliferate to repopulate the tubule when cells are lost due to
damage and cell death.
In some embodiments, the result of acute kidney injury is EMT. EMT
is a process by which epithelial cells lose their cell polarity and
cell-cell adhesion, and gain migratory and invasive properties to
become mesenchymal stem cells, which can either remain mesenchymal
or differentiate back to epithelial cells. EMT can occur in wound
healing, in tissue fibrosis, and in the initiation of metastasis
for cancer progression.
In some embodiments, the result of acute kidney injury is
inflammation. In some embodiments, acute kidney injury can be
caused by inflammation. In some embodiments, the inflammation that
causes or results from acute kidney injury is caused by the
activation of pattern recognition receptors. In some embodiments,
the inflammation that causes or results from acute kidney injury is
caused by cytokines. In some embodiments, the inflammation that
causes or results from acute kidney injury is caused by
chemokines.
In some embodiments, the result of acute kidney injury is ischemia.
In some embodiments, activation of hypoxia-inducible transcription
factor (HIF) protects against ischemia. HIF has been identified as
an important mechanism of cellular adaptation to low oxygen
(hypoxia).
In some embodiments, the result of acute kidney injury is a change
in the mitochondria. Changes in the mitochondria caused by acute
kidney injury include changes in mitochondrial glutathione levels,
changes in reactive oxygen species, and changes in mitochondrial
morphology. In some embodiments, the result of acute kidney injury
is mitochondrial impairment. Mitochondrial impairment includes loss
of mitochondrial membrane potential, reduction in mitochondrial
biogenesis, and a drop in ATP production (Granata et al.,
2015).
In some embodiments, the result of acute kidney injury is a change
to cell morphology. Changes in cell morphology caused by acute
kidney injury include changes in the amount of cytoplasm and
changes in the shape of the cell.
In some embodiments, such as with hyperglycemia, the result of
proximal tubule cell injury is evidenced by a change in nuclear
morphology. In some embodiments, the cellular injury presents as
the accumulation of glycogen. In some embodiments, glycogen
accumulates in the nucleus, such that the nuclei appear clear and
vacuolated in standard histological stains such as H&E and
Periodic Acid Shiff. In other embodiments, cellular injury is
evidenced by accumulation of glycogen in the cytoplasm.
In some embodiments, the result of acute kidney injury is
hyperproliferation. Hyperproliferation is an abnormally high rate
of proliferation by cells by rapid division.
In some embodiments, the result of acute kidney injury is an
alteration in gene expression. In some embodiments, the alteration
of gene expression may lead to downstream changes in protein
expression and/or function.
In some embodiments, the result of acute kidney injury is a
secretion of biomarkers. Biomarkers of acute kidney injury can be
components of serum or urine or can be imaging studies. In some
embodiments, the biomarkers of acute kidney injury include
N-acetyl-.beta.-glucosamide, .beta..sub.2-microglobulin,
.alpha..sub.1-microglobulin, retinol binding protein, cystatin-C,
microalbumin, kidney injury molecule-1, clusterin, neutrophil
gelatinase-associated lipocalin, interleukin-18, cysteine-rich
protein, osteopontin, fatty acid-binding protein, sodium/hydrogen
exchanger isoform, or fetuin-A (Vaidya et al., 2008).
In some embodiments, the result of acute kidney injury is an
epigenetic modification. Epigenetics refers to the modulation of
gene expression via post-translational modification of protein
complexes which are associated with DNA but do not change the DNA
sequence such as acetylation, methylation, phosphorylation,
ubiquitinylation, sumoylation, carbonylation, glycosylation, and
expression of microRNA (Tang et al., 2015). In some embodiments,
the epigenetic modification may lead to downstream changes in
protein expression and/or function.
Chronic Renal Disorder
In some embodiments, the renal disorder is a chronic renal
disorder. Chronic renal disorder is also called chronic kidney
disease or chronic kidney failure. In some embodiments, the chronic
renal disorder is chronic kidney injury. Chronic kidney injury is
the progressive deterioration of renal function.
In some embodiments, tubular flow is required to develop a relevant
phenotype characteristic of a chronic renal disorder.
In some embodiments, chronic renal disorder is caused by the same
mechanisms as acute renal disorder but with exposure over a longer
period of time. In some embodiments, the chronic kidney injury is
caused by toxicant exposure, diabetes, infection, inflammation,
ischemia, crystal deposition, a genetic disorder, a cystic disease,
a chronic system disorder, or a transport deficiency.
In some embodiments, the chronic kidney injury is caused by
toxicant exposure. In some embodiments, the toxicant is an
anti-infective. Anti-infectives include antibiotics,
antibacterials, antifungals, and antiviral. In some embodiments,
the toxicant is an antibiotic, an antibacterial, an antifungal, or
an antiviral. In some embodiments, the toxicant is acetaminophen,
lithium, acyclovir, amphotericin B, an aminoglycoside, a beta
lactam, foscavir, ganciclovir, pentamidine, a quinolone, a
sulfonamide, vancomycin, rifampin, adefovir, indinavir, didofovir,
tenofovir, methotrexate, lansoprazole, omeprazole, pantopraxole,
allopurinol, phenytoin, ifosfamide, gentamicin, or zoledronate.
In some embodiments, the chronic kidney injury is secondary to
diabetes. In some embodiments, the diabetes is type 2 diabetes. In
some embodiments, the diabetes is type 1 diabetes. In some
embodiments, the chronic kidney disease is secondary to
hypertension and vascular/glomerular injury. In some embodiments,
the proximal tubule injury is secondary to a decline in glomerular
integrity and function, which leads to elevated glucose, proteins
such as albumin, or other blood components in the filtrate. In some
embodiments, the kidney injury resulting from diabetes is caused in
part by exposure of the tubule to elevated levels of glucose,
protein, and other blood components in the filtrate. In some
embodiments, the acute kidney injury is caused by DKA. In some
embodiments, tubular injury is further compromised by loss or
compromise of microvasculature and resulting hypoxia.
In some embodiments, the chronic kidney injury is caused by
infection. In some embodiments, the infection is caused by a
microorganism or microbe. In some embodiments, the microorganism
that causes infection is a bacteria, a virus, a fungi, a protozoa,
or a helminth.
In some embodiments, the chronic kidney injury is caused by
inflammation. In some embodiments, the inflammation is caused by a
pattern recognition receptor. Pattern recognition receptors include
toll-like receptors (TLRs), retinoic acid-inducible gene
(RIG)-I-like receptors, NOD-like receptors, and C-type lectin
receptors. In some embodiments, the inflammation is caused by a
TLR, a RIG-I-like receptor, a NOD-like receptor, or a C-type lectin
receptor. In some embodiments, the inflammation that causes chronic
kidney injury is caused by cytokines. In some embodiments, the
inflammation that causes chronic kidney injury is caused by
chemokines.
In some embodiments, the chronic kidney injury is caused by
persistent ischemia.
In some embodiments, the chronic kidney injury is caused by crystal
deposition. Crystal deposition in the kidney can result from
different mechanisms including: (1) Crystal embolism, mostly caused
by cholesterol crystals originating from atherosclerotic lesions of
the aorta. These crystals can obstruct smaller arteries and
arterioles and lead to ishemic kidney injury. (2) Intratubular cast
formation leading to obstruction of distal tubules. (3) Diffuse
crystallization with intratubular plugs and intratubular and
intrastitial crystals like in oxalate nephropathy or cystinosis.
(4) Pelvic stone formation at the papilla, which consist of calcium
phosphate precipitates in the interstitium at the thin loop of
Henle. The resulting lesion (Randall's plaque) becomes an
attachment site for the precipitation of other urinary crystals
that can grow to stones. (Mulay et al., 2014). In some embodiments,
the crystal or particle is cholesterol monosodium urate, calcium
oxalate, calcium phosphate hydroxyapatite, 2,8-dihydroxyadenine,
uromodulin, myoglobin-uromodulin, indinavir, acyclovir, a polymyxin
(e.g., polysporin, neosporin, polymyxin B, or polymyxin E),
sulfadiazine, cysteine, uric acid, or magnesium ammonium
phosphate.
In some embodiments, the chronic kidney injury is caused by a
genetic disorder.
In some embodiments, the genetic disorder is a cystic kidney
disease, Alport's syndrome, Bartter's syndrome, cystinosis,
cystinuria, hyperoxaluria, congenital nephrotic syndrome,
nail-patella syndrome, primary immune glomerulonephritis, reflux
nephropathy, or haemolytic uraemic syndrome. In some embodiments,
the genetic disorder is a cystic kidney disease such as autosomal
dominant polycystic kidney disease, autosomal recessive polycystic
kidney disease, juvenile nephronophthiasis, adult nephronophthisis,
medullary sponge kidney, a cystic kidney disease associated with a
multiple malformation syndrome (e.g., tuberous sclerosis, Lowe's
syndrome, or Von Hippel-Lindau disease).
In some embodiments, the chronic kidney injury is caused by a
chronic system disorder such as diabetes, an autoimmune disease
(e.g., systemic lupus erythematosus or Goodpasture's syndrome), or
gout.
In some embodiments, the chronic kidney injury is caused by a
transport deficiency. In some embodiments, the chronic kidney
injury is caused by a glucose transport deficiency.
In some embodiments, the chronic kidney injury is caused by an
accumulation of proteins, salts, or other precipitous matter.
In some embodiments, the result of chronic kidney injury is
necrosis, apoptosis, nephritis, tubular regeneration, EMT,
inflammation, ischemia, changes in the mitochondria, changes to
cell morphology, changes to nuclear morphology, hyperproliferation,
alternations in gene expression, secretion of biomarkers,
epigenetic modifications, or crystal deposition.
In some embodiments, the result of chronic kidney injury is
necrosis. In some embodiments, chronic kidney injury caused by
exposure to toxicants results in necrosis.
In some embodiments, the result of chronic kidney injury is
apoptosis. In some embodiments, chronic kidney injury caused by
exposure to toxicants results in apoptosis.
In some embodiments, the result of chronic kidney injury is tubular
regeneration.
In some embodiment, the result of chronic kidney injury is
compensatory proliferation.
In some embodiments, the result of chronic kidney injury is
EMT.
In some embodiments, the result of chronic kidney injury is
inflammation. In some embodiments, chronic kidney injury can be
caused by inflammation. In some embodiments, the inflammation that
results from chronic kidney injury is caused by the activation of
pattern recognition receptors. In some embodiments, the
inflammation that results from chronic kidney injury is caused by
cytokines. In some embodiments, the inflammation that results from
chronic kidney injury is caused by chemokines.
In some embodiments, the result of chronic kidney injury is
ischemia. In some embodiments, activation of hypoxia-inducible
transcription factor (HIF) protects against ischemia.
In some embodiments, the result of chronic kidney injury is a
change in the mitochondria. Changes in the mitochondria caused by
chronic kidney injury include changes in mitochondrial glutathione
levels, changes in reactive oxygen species, and changes in
mitochondrial morphology. In some embodiments, the result of
chronic kidney injury is mitochondrial impairment. Mitochondrial
impairment includes loss of mitochondrial membrane potential,
reduction in mitochondrial biogenesis, and a drop of ATP production
(Granata et al., 2015).
In some embodiments, the result of chronic kidney injury is a
change to cell morphology. Changes in cell morphology caused by
chronic kidney injury include changes in the amount of cytoplasm,
changes in the shape of the cell and cyst formation.
In some embodiments, the result of proximal tubule cell injury is
evidenced by a change in nuclear morphology. In some embodiments,
the cellular injury presents as the accumulation of glycogen. In
some embodiments, glycogen accumulates in the nucleus, such that
the nuclei appear clear and vacuolated in standard histological
stains such as H&E and Periodic Acid Shiff. In other
embodiments, cellular injury is evidenced by accumulation of
glycogen in the cytoplasm.
In some embodiments, the result of chronic kidney injury is
hyperproliferation.
In some embodiments, the result of chronic kidney injury is an
alteration in gene expression. In some embodiments, the alteration
of gene expression may lead to downstream changes in protein
expression and/or function.
In some embodiments, the result of chronic kidney injury is a
secretion of biomarkers. Biomarkers of chronic kidney injury can be
components of serum or urine or can be imaging studies. In some
embodiments, the biomarkers of chronic kidney injury include
N-acetyl-.beta.-glucosamide, .beta..sub.2-microglubulin,
.alpha..sub.1-microglobulin, retinol binding protein, cystatin-C,
microalbumin, kidney injury molecule-1, clusterin, neutrophil
gelatinase-associated lipocalin, interleukin-18, cysteine-rich
protein, osteopontin, fatty acid-binding protein, sodium/hydrogen
exchanger isoform, or fetuin-A (Vaidya et al., 2008).
In some embodiments, the result of chronic kidney injury is an
epigenetic modification. Epigenetic modification include
modifications to the nucleotides or DNA backbone by acetylation,
methylation, phosphorylation, ubiquitinylation, sumoylation,
carbonylation, glycosylation, or expression of microRNA. In some
embodiments, the epigenetic modification may lead to downstream
changes in protein expression and/or function.
In some embodiments, the result of chronic kidney injury is the
presence of a crystal or a particle. In some embodiments, the
crystal or particle is cholesterol monosodium urate, calcium
oxalate, calcium phosphate hydroxyapatite, 2,8-dihydroxyadenine,
uromodulin, myoglobin-uromodulin, indinavir, acyclovir, a polymyxin
(e.g., polysporin, neosporin, polymyxin B, or polymyxin E),
sulfadiazine, cysteine, uric acid, or magnesium ammonium
phosphate.
RENAL CANCER
In some embodiments, the renal disorder is a renal cancer.
In some embodiments, the renal cancer is renal cell carcinoma,
transitional cell carcinoma, Wilms' tumor, or renal sarcoma. In
some embodiments, the renal cancer is a renal cell carcinoma such
as clear cell renal cell carcinoma, papillary renal cell carcinoma,
chromophobe renal cell carcinoma, collecting duct renal cell
carcinoma, multilocular cystic renal cell carcinoma, medullary
carcinoma, mucinous tubular and spindle cell carcinoma, or
neuroblastoma-associated renal cell carcinoma.
In some embodiments, the renal cancer is caused by protein
modifications, gene mutations, gene translocations, chemical
exposure, genetic dysfunction, or an epigenetic modification.
In some embodiments, the renal cancer is caused by protein
modifications. In some embodiments, the protein modification is
phosphorylation of a protein or truncation of a protein.
In some embodiments, the renal cancer is caused by gene mutations.
In some embodiments, the gene mutation turns on an oncogene or
turns off a tumor suppressor gene. In some embodiments, the gene
mutation is an inherited gene mutation such as a mutation in the
VHL gene, a mutation in the FH gene, a mutation in the FLCN gene, a
mutation in the SDHB gene, a mutation in the SDHD gene, or a
mutation in the MET oncogene. In some embodiments, the gene
mutation is an acquired gene mutation such as a mutation in the
tumor suppressor gene and/or oncogene caused by cancer-causing
chemicals. In some embodiments, the gene mutation is an acquired
gene mutation caused by a mutation to the VHL gene. In some
embodiments, the alteration of gene expression may lead to
downstream changes in protein expression and/or function.
In some embodiments, the renal cancer is caused by gene
translocations. Gene translocation can lead to fusion between two
different genes that results in a protein with altered function
(i.e., BCR-ABL gene fusion).
In some embodiments, the renal cancer is caused by exposure to a
chemical that leads to renal cancer.
In some embodiments, the renal cancer is caused by a genetic
dysfunction. In some embodiments, the genetic dysfunction causes a
mutation in the nucleotide. In some embodiments, the mutation may
lead to downstream changes in protein expression and/or
function.
In some embodiments, the renal cancer is caused by an epigenetic
modification.
Epigenetic modification includes modifications to the nucleotides
or DNA backbone by acetylation, methylation, phosphorylation,
ubiquitinylation, sumoylation, carbonylation, glycosylation, or
expression of microRNA. In some embodiments, the epigenetic
modification may lead to downstream changes in protein expression
and/or function.
In some embodiments, the result of renal cancer is
hyperproliferation, angiogenesis, hypoxia, or death of surrounding
tissue.
In some embodiments, the result of renal cancer is
hyperproliferation.
In some embodiments, the result of renal cancer is
angiogenesis.
In some embodimens, the result of renal cancer is hypoxia. Tumor
hypoxia is the situation where tumor cells have been deprived of
oxygen. As a tumor grows, it rapidly outgrows its blood supply,
leaving portions of the tumor with regions where the oxygen
concentration is significantly lower than in healthy tissues.
In some embodiments, the result of renal cancer is the death of
surrounding tissue.
Methods of Producting a Renal Disorder in the Renal Tubule
Model
In some embodiments, a renal disorder as described herein is
produced in a renal tubule model as described herein by contacting
the model with a molecule such as a toxicant, or a high level of
glucose to produce a renal tubule phenotype that is characetistic
of the renal disorder.
In some embodiments, a renal disorder is produced in the renal
tubule model by genetically modified cells in the model. In some
embodiments, the genetically modified cells are modified before the
model is formed. In some embodiments, the genetically modified
cells are modified after the model is formed. In some embodiments,
the genetic modification is a polycystic mutation in a transporter.
In some embodiments, the genetic modification is made by using a
retrovirus, CRISPR, viral transduction, or chemical mutagenesis. In
some embodiments, the genetic modification is made in a stem cell,
which is then used to fabricate the renal tubule disorder model. In
some embodiments, the bio-ink includes a stem cell having the
genetic modification, with the bio-ink being used to fabricate the
renal tubule disorder model.
In some embodiments, the renal disorder is produced in the renal
tubule model by using cells from diseased donors and using them as
cell inputs. In some embodiments, cells can be isolated from donors
with a specific disease and used to fabricate the renal tubule
disorder model. In some embodiments, the bio-ink includes cells
isolatd from donors with a specific disease, with the bio-ink being
used to fabricate the renal tubule disorder model. In other
embodiments, induced pluripotent stem cells can be taken from
adults with a genetic dysfunction and used to fabricate the renal
tubule disorder model. In some embodiments, the bio-ink includes
induced pluripotent stem cells taken from adults with a genetic
dysfunction, with the bio-ink being used to fabric the renal tubule
disorder model.
In some embodiments, the fibroblasts, endothelial cells, and/or
epithelial cells can be genetically modified prior to incorporation
into the tissue or after tissue formation to induce the disease
phenotype. In some embodiments, the bio-ink includes genetically
modified fibroblasts, endothelial cells, epithelial cells, or other
kidney cells, with the bio-ink being used to fabricate the renal
tubule disorder model.
Testing the Viability or Functionality of the Renal Tubule
Model
In some embodiments, the viability or functionality of the renal
tubule model is determined by measuring the induction of an
apoptotic pathway. In some embodiments, the viability or
functionality of the renal tubule model is determined by caspase
activation. In some embodiments, caspase activity is measured using
synthetic peptide substrates (Kumar, 2004). In other embodiments,
the viability or functionality of the renal tubule model is
determined by measuring the hallmarks of apoptosis such as
chromatin condensation, nuclear fragmentation, or mitochondrial
release or cytochrome c.
In some embodiments, the viability or functionality of the renal
tubule model is determined by measuring changes in cellular or
nuclear morphology. Methods of measuring changes in cellular or
nuclear morphology include examination by histology or
microscopy.
In some embodiments, the viability or functionality of the renal
tubule model is determined by measuring changes in the number or
morphology of mitochondria. The number and morphology of
mitochondria can be measured using histology or microscopy. In some
embodiments, the viability or functionality of the renal tubule
model is determined by measuring the downstream function of
mitochondria. The downstream function of mitochondria can be
measured using a commercially available kit to measure mitochondria
respiration.
In some embodiments, the viability or functionality of the renal
tubule model is determined by measuring the secretion of a cytokine
or a chemokine. In some embodiments, the secretion of cytokines and
chemokines can be measured by histology, ELISA, mass spectroscopy,
a clinical chemical analyzer, or an immunoassay analyzer.
In some embodiments, the viability or functionality of the renal
tubule model is determined by measuring the amount and/or pattern
of deposition of the extracellular matrix. In some embodiments, the
amount and/or pattern of deposition of the extracellular matrix can
be measured by histology or an immunoassay.
In some embodiments, the viability or functionality of the renal
tubule model is determined by measuring the deposition of a protein
crystal or a salt crystals within the tissue. In some embodiments,
the deposition of a protein crystal or a salt crystal within the
tissue can be measured using histology or microscopy.
In some embodiments, the viability or functionality of the renal
tubule model is determined by measuring tubular regeneration or
compensatory proliferation. In some embodiments, tubular
regeneration or compensatory proliferation can be measured using
histology. In some embodiments, the histological stain is for
proliferating cell nuclear antigen (PCNA) or Ki-67.
In some embodiments, the viability or functionality of the renal
tubule model is determined by measuring epithelial-mesenchymal
transition (EMT). In some embodiments, EMT can be measured using
histology or microscopy.
In some embodiments, the viability or functionality of the renal
tubule model is determined by measuring inflammation. In some
embodiments, inflammation can be measured using histology, ELISA,
mass spectroscopy, a clinical chemical analyzer, an immunoassay
analyzer, or by gene expression using a molecular diagnostic
analyzer.
In some embodiments, the viability or functionality of the renal
tubule model is determined by measuring ischemia. In some
embodiments, ischemia can be measured by looking for the evidence
of hypoxia inductable factors. Evicence of hypoxia inductable
factors can be measured using histology or by gene expression using
a molecular diagnostic analyzer.
In some embodiments, the viability or functionality of the renal
tubule model is determined by measuring hyperproliferation. In some
embodiments, hyperproliferation can be measured using histology or
microscopy.
In some embodiments, the viability or functionality of the renal
tubule model is determined by measuring alterations in gene
expression. In some embodiments, alterations in gene expression can
be measured using a molecular diagnostic analyzer (e.g., a
microarray, RNA sequencing, or a qPCR).
In some embodiments, the viability or functionality of the renal
tubule model is determined by alterations in protein expression
and/or post-translational modification. In some embodiments, levels
of protein expression and post-translational modification of
proteins can be measured using histology, microscopy, flow
cytometry, Western blot, ELISA, an immunoassay, or a molecular
diagnostic analyzer.
In some embodiments, the viability or functionality of the renal
tubule model is determined by measuring secretion of biomarkers. In
some embodiments, secretion of biomarkers can be measured using
ELISA, a protein activity assay, an immunoassay, or a molecular
diagnostic analyzer. In other embodiments, secretion of nuclear
biomarkers such as micro-RNAS can be measured using a molecular
diagnostic analyzer (e.g., a microarray, RNA sequencing, or a
qPCR). In other embodiments, secretion of other chemical biomarkers
can be measured using mass spectroscopy or a clinical chemical
analyzer.
In some embodiments, the viability or functionality of the renal
tubule model is determined by measuring epigenetic changes. In some
embodiments, epigenetic changes can be measured using a molecular
diagnostic analyzer (e.g., a DNA methylation kit).
In some embodiments, the viability or functionality of the renal
tissue cells is determined by measuring changes in expression
and/or concentration of cytoplasmic proline-rich tyrosine kinase-2
(Pyk2) expression, thiazide-sensitive cotransporter (TSC)
expression, epidermal growth factor (EGF) expression, transforming
growth factor-alpha (TGF-.alpha.) expression, stem cell factor
(SCF) expression, transforming growth factor-beta (TGF-.beta.)
expression, connective growth tissue factor (CTGF) expression,
complement factor B expression, toll-like receptor 2 (TLR2)
expression, toll-like receptor 4 (TLR4) expression, interleukin-6
(IL-6) expression, Class II major histocompatibility complex (MEW)
expression, intercellular adhesion moleculare-1 (ICAM-1)
expression, monocyte chemoattractant protein-1 (MCP-1) expression,
or plasminogen activator inhibitor-1 (PAI-1) expression compared to
a control. Changes in expression and/or concentration of these
factors may be measured according to methods that are well known in
the art including antibody based assays.
Cytoplasmic proline-rich tyrosine kinase-2 (Pyk2) has been found to
be abundantly expressed in tubular epithelial cells were it is
activated by several stimuli including agonists for G
protein-coupled receptors, intracellular calcium concentration,
inflammatory cytokines, stress signals, and integrin-mediated cell
adhesion (Sonomura et al., 2012). It is believed that Pyk2 may be
an important initiating factor in renal fibrosis.
Thiazide-sensitive cotransporter (TSC) has been shown to be
localized to the distal convoluted tubule in the kidney by in situ
hybridization studies, in reverse transcription, and in polymerase
chain reaction with microdissected nephron segments (Taniyama et
al., 2001). Mutations that may lead to loss of function in the
human TSC gene have been shown to cause Gitelman's syndrome, which
is characterized by dehydration, hypokalemic metabolic alkalosis,
hypomagnesemia, and hypocalciuria.
ErbB signaling has been found to be involved in renal electrolyte
homeostatis and maintenance of kidney integrity (Melenhorst et al.,
2008). The ErbB receptor family belongs to subclass I of the
receptor tyrosine kinase superfamily and incorporates epidermal
growth factor (EGF) receptors (EGFR, HER1, and ErbB1), HER2/neu
(ErbB2), HER3 (ErbB3), and HER4 (ErbB4). EGFR expression has been
detected in the tubules in most normal human kidneys. Furthermore,
an increase in EGF was associated with a decrease in renal function
and decreased tubulointerstitial EGF expression correlated with the
severity of apoptosis.
Transforming growth factor-alpha (TGF-.alpha.) has been detected in
primitive tubules in human kidney dysplasia (Melenhorst et al.,
2008).
The cytokine stem cell factor (SCF) has been shown to protect the
tubular epithelium against apoptosis (Stokman, G., 2010). Survival
of the tubular epithelium is important to successfully regenerate
renal tissue following renal ischemia.
Transforming growth factor-beta 1 (TGF-.beta.1) has been found to
promote tissue regeneration following acute injury via an autocrine
or paracrine mechanism (Basile et al., 1996). Elevated expression
of TGF-.beta.1 was found to be localized predominantly to cells in
the regenerating tubules in the outer medulla. TGF-.beta.1
expression was also found to be inhibited by peroxisome
proliferator-activated receptor-.gamma.(PPAR-.gamma.) (Wang et al.,
2009). PPAR-.gamma. has been found to have anti-inflammatory
effects in kidney disease.
Connective tissue growth factor (CTGF) has been found to act as a
downstream mediator for the profibrotic effects of TGF-.beta.1 in
the remant kidney and may be a target for antifibrotic drugs
designed to treat TGF-.beta.1 dependent interstitial fibrosis
(Okada et al., 2005). It has also been found that after treatment
with the glucocorticoid dexamethasone, renal tubular epithelial
cells from patients with minimal change nephritic syndrome produced
CTGF (Okada et al., 2006).
The expression of complement factor B has been shown to increase in
human proximal tubular cells and mouse tubular epithelial cells
after stimulation with toll-like receptor 4 (lipopolysaccharide) or
toll-like receptor 3 (polyinosinic-olycytidylic acid) (Li et al.,
2016).
Exposure of renal tubular epithelial cells to tumor necrotic factor
alpha (TNF-.alpha.) and triptolide followed by examination of
expression of B7-H1 and B7-DC by flow cytometric analysis, showed
that B7-H1 but not B7-DC constitutively expresses on renal tubular
epithelial cells (Chen et al., 2006). And, B7-H1 was shown to be
profoundly upregulated by the stimulation of TNF-.alpha.and
downregulated by triptolide. A distinct expression pattern of
toll-like receptors (TLRs) was found in mouse primary renal tubular
epithelial cells and it was found that the epithelial cells
secreted C-C chemokines in response to direct stimulation (Tsuboi
et al., 2002). In particular, it was shown that TLR2 and TLR4
expressed in mouse primary renal tubular epithelial cells mediated
direct responses to bacterial components.
Interleukin-6 (IL-6) expression in renal tubular epithelial cells
has been found to be inhibited by administration of the
immunosuppressant drug mycophenolic acid (Baer et al., 2004). IL-6
has been implicated in the development of tubular injury in various
forms of immune-mediated renal diseases.
Class II major histocompatibility complex (MHC) and B7-1 expression
in renal tubular epithelial cells were found to be mediated by
interferon-gamma (IFN-y) and liposaccharide (Banu et al.,
1999).
Intercellular adhesion molecular-1 (ICAM-1) expression was found to
be upregulated in renal tubular epithelial cells by the cytokines
interferon-.gamma., TNF-.gamma., and IL-1 (Ishikura et al.,
1991).
Monocyte chemoattractant protein-1 (MCP-1), a chemokine with potent
chemotactic activity for monocytes/macrophages and T lymphocytes,
has been found to be upregulated in proximal renal tubular cells
challenged with protein overload (Zoja, et al., 2003). And, the
chemokine fractalkine was also found to be overexpressed upon
albumin stimulation of proximal renal tubular cells.
Angiotensin II and Angiotensin IV were shown to induce an increase
in plasminogen activator inhibitor-1 (PAI-1) expression in a
proximal tubular epithelial cell line from a normal adult human
kidney (Gesualdo et al., 1999). PAI-1 has been found to prevent the
transformation of metalloproteinases, which are potent ECM
degradation enzymes, which contribute to tubulointerstitial
fibrosis.
Models of Renal Fibrosis
Provided are models of renal fibrosis, comprising a
three-dimensional, engineered, bioprinted, biological renal tubule
model. In some embodiments, the three-dimensional, engineered,
bioprinted, biological renal tubule models comprise a layer of
renal interstitial tissue, the renal interstitial tissue comprising
renal fibroblasts, endothelial cells and/or fibrotic tissue; and a
layer of renal epithelial tissue, the renal epithelial tissue
comprising renal tubular epithelial cells; provided that the
interstitial tissue comprises an interstitial bio-ink, the
epithelial tissue comprises an epithelial bio-ink, and form a
three-dimensional, engineered, biological renal tubule model. In
one embodiment, the model of renal fibrosis displays contraction,
curling, expansion of the tissue, or another fibrosis phenotype
when fibrosis is present in the model.
In some embodiments, the model further comprises a layer of
basement membrane between the renal interstitial tissue layer and
the renal epithelial tissue layer. In some embodiments, the layer
of renal epithelial tissue is in continuous contact with the layer
of basement membrane, and the layer of basement membrane is in
continuous contact with the layer of renal interstitial tissue.
In some embodiments, the fibroblasts and endothelial cells are
present in the layer of renal interstitial tissue at a ratio of
about 50:50 fibroblasts to endothelial cells.
In some embodiments, the renal tubular model displays deformation
of the planar tissue structure and excess extracellular matrix
deposition.
In some embodiments, the fibroblasts and endothelial cells are
present in a ratio at which the renal tubule model is planar six
days post-printing.
Also provided are methods of making the model of renal fibrosis
comprising contacting a three-dimensional, engineered, bioprinted,
biological renal tubule model with an agent that is capable of
inducing interstitial fibrotic tissue formation, wherein the renal
tubule model comprises: a layer of renal interstitial tissue, the
renal interstitial tissue comprising renal fibroblasts and
endothelial cells; and a layer of renal epithelial tissue, the
renal epithelial tissue comprising renal tubular epithelial cells,
to form the three-dimensional, engineered, biological renal tubule
model; provided that the interstitial tissue comprises an
interstitial bio-ink, the epithelial tissue comprises an epithelial
bio-ink, and form a three-dimensional, engineered, biological renal
tubule model.
In some embodiments, the agent that is capable of inducing
interstitial fibrotic tissue deposition is cyclosporine A,
aristolochoic acid, tacrolimus, TGF-.beta., cisplatin, acyclovir,
allopurinol, beta lactam antibiotics, indinavir, lansoprazole,
omeprazole, pantoprazole, phenytoin, ranitidine, or vancomycin.
The disclosure herein includes business methods. In some
embodiments, the speed and scalability of the techniques and
methods disclosed herein are utilized to design, build, and operate
industrial and/or commercial facilities for production of renal
tubule models for use in cell-based tools for research and
development, such as in vitro assays. In further embodiments, the
renal tubule models and arrays thereof are produced, stored,
distributed, marketed, advertised, and sold as, for example,
cellular arrays (e.g., microarrays or chips), tissue arrays (e.g.,
microarrays or chips), and kits for biological assays and
high-throughput drug screening. In other embodiments, the
engineered renal tubule models and arrays thereof are produced and
utilized to conduct biological assays and/or drug screening as a
service.
Validation
The ideal engineered renal tissues are fully human and
multicellular, comprising renal tubular epithelial cells, renal
interstitial fibroblasts, and endothelial cells. Moreover, ideal
engineered renal tissues demonstrate specific functions including,
but not limited to, CYP1A2, CYP2C9, and CYP3A4 activity, albumin
transport, and vitamin D hydroxylation,
.gamma.-glutamyl-transferase activity. Also, the ideal engineered
renal tissues are characterized by tight junctions, cadherin,
polarity of transporters, and CD31 expression and are validated by
specific assays including albumin transport, CYP450 activity,
histology, and viability. In some embodiments, the renal tubule
models of the present disclosure display increased specific
functions compared to 2D co-culture or tissue explants that have
been maintained in culture longer than 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, or more days. In some embodiments, the renal tubule models of
the present disclosure display 2-fold increased specific functions
compared to 2D co-culture or tissue explants that have been
maintained in culture longer than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or
more days. In some embodiments, the renal tubule models of the
present disclosure display 5-fold or more increased specific
functions compared to 2D co-culture or tissue explants that have
been maintained in culture longer than 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, or more days. In some embodiments, the renal tubule models of
the present disclosure display 2-fold or more increased specific
functions compared to 2D co-culture or tissue explants that have
been maintained in culture longer than 21 or more days. In some
embodiments, the renal tubule models of the present disclosure
display 5-fold or more increased specific functions compared to 2D
co-culture or tissue explants that have been maintained in culture
longer than 27 or more days. In some embodiments, the renal tubule
models of the present disclosure display 2-fold or more increased
specific functions compared to 2D co-culture or tissue explants
that have been maintained in culture longer than 27 or more days.
In some embodiments, the renal tubule models of the present
disclosure display 5-fold or more increased specific functions
compared to 2D co-culture or tissue explants that have been
maintained in culture longer than 21 or more days. In certain
embodiments, the specific function is .gamma.-glutamyl-transferase
activity. In certain embodiments, the specific function is vitamin
D hydroxylation.
In some embodiments, the engineered tissues described herein
possess key architectural and functional attributes associated with
in vivo human renal tissue, including histologic features and renal
tubule-specific functions, including but not limited to:
Polarization of renal tubular epithelial cells w/formation of
intracellular tight junctions (E-Cad, ZO-1, and Claudins) and
correct intracellular localization of transporters (apical: OAT4,
URAT1) and integrins (basolateral). Development of a basal lamina
between the tubular cell layer and the underlying interstitium.
Establishment of extensive microvascular networks within the
interstitium, including the development of tissue-like tubular
cells: microvascular spatial relationships. Expression of
compartment-specific markers, including tubular epithelial
transporters (cubilin, megalin, aquaporins), OATs, URAT), vascular
markers (CD31, vWF), demonstration of EPO protein production (if
applicable). Vitamin D synthesis via 25-(OH) 1-hydroxylase
(1-OHase). Production of Angiotensin II. Active transport of
albumin from tubular lumen via cubilin. Cimetidine
transport/accumulation from basolateral surface. CYP450 and UGT
expression involved in metabolism (e.g., CYP2B6, 3A5, 4A11 and UGT
1A9, 2B7, respectively).
EXAMPLES
The following illustrative examples are representative of
embodiments, of the software applications, systems, and methods
described herein and are not meant to be limiting in any way.
Example 1
A Bioprinted Three-Dimensional Renal Tubule Cell Model
Human umbilical vein endothelial cells (HUVEC) were purchased from
BD
Biosciences (Franklin Lakes, N.J.) and cultured in EGM-2 media with
EBM-2 supplements without gentamycin or amphotericin B (Lonza,
Basel, Switzerland). Adult renal fibroblasts were purchased from DV
Biologics (Yorba Linda, Calif.) and grown in Fibroblast Cellutions
Medium with Fibroblast Cellutions supplement (DV Biologics, Yorba
Linda, Calif.). Primary human RPTEC were purchased from four
different commercial vendors (Lonza, Sciencell (Carlsbad, Calif.),
Zen-Bio (Research Triangle Park, N.C.), Lifeline Cell Technology
(Frederick, Md.) and cultured according to the manufacturer's
instructions.
All kidneys were ethically sourced through the National Disease
Research Interchange (Philadelphia, Pa.). RPTEC cells were isolated
as previously described (Vesey et al., 2009). In brief, upon
receipt, kidneys were aseptically unpacked and cleaned to remove
any remaining fat pads, ureters, blood vessels or other tissue.
Sections of cortical tissue were minced, digested with collagenase,
and the collected cells were enriched for epithelium by
centrifugation across an iodixanol gradient (Sigma-Aldrich, St.
Louis, Mo.). RPTECs were cultured in GBG.TM. Epithelial Media
(Samsara Sciences, San Diego, Calif.).
Example 2
A Bioprinted Three-Dimensional Renal Tubule Cell Model
3D PT Novo View.TM. Tissues were fabricated as described (Nguyen et
al., 2016). Briefly, cultured renal fibroblasts and HUVEC were
combined in a 50:50 ratio and resuspended in NovoGel.RTM. Bio-Ink,
and then bioprinted onto 0.4 .mu.m Transwell.RTM. clear polyester
membrane inserts in a 24-well plate (Coming Costar, Coming, NY)
using a NovoGen Bioprinter.RTM. Instrument (Organovo Inc., San
Diego, Calif.) with previously established protocols (Forgacs et
al., 2012; Murphy et al., 2015; Nguyen et al., 2016). Following
bioprinting, NovoView' Tissues were cultured in NovoView.TM. Kidney
Media (Organovo, San Diego, Calif.). On culture day 3, primary
RPTEC cells were added to the tissues in a suspension of
1.25.times.10.sup.6 cells/ml in RPTEC media. Tissues were then
maintained for up to 30 days in Novo View.TM. Kidney Media
(Organovo, San Diego, Calif.), with media exchanges every other
day. For toxicity studies, tissues were dosed daily to the apical
and basolateral compartments beginning at day 14 of culture. For
cisplatin dosing studies, culture media was supplemented with a
final concentration of 2.5% FBS v/v to the apical and basolateral
compartments of the Transwell.RTM. inserts.
Example 3
Metabolic and Viability Assays on Bioprinted Tissues
Assessment of metabolic activity as a surrogate for tissue
viability and health was performed by alamarBlue.TM. assay
according to the manufacturer's protocol (Thermo Fisher, Carlsbad,
Calif.). Briefly, tissues were washed twice with Dulbecco's
phosphate buffered saline (DPBS), and RPTEC media supplemented with
10% v/v alamarBlue' reagent was added to each tissue. All tissues
were incubated for 2 hours at 37.degree. C. with 95% relative
humidity and 5% CO2. After incubation, the alamarBlue' solution was
removed and fluorescence was measured on a BMG Labtech
POLARstar.RTM. Omega reader (Cary, N.C.) with an excitation filter
of 560 nm and an emission filter of 590 nm. Graphed data represent
the percent relative fluorescence units (RFU) compared to blank for
metabolic activity over time, or the percent RFU compared to
vehicle control for toxicity studies.
Lactate dehydrogenase (LDH) activity assay was performed according
to the manufacturer's protocol (Abcam, Cambridge, Mass.).
Conditioned media was collected from 3D PT tissues and further
diluted in fresh media to ensure that the LDH activity of the
sample was within the linear range of the assay. Samples were
measured on a microplate reader (BMG Labtech, Cary, N.C.). LDH
activity was determined by standard curve integration of absorbance
normalized for volume and duration using GraphPad Prism.RTM.
software (GraphPad, San Diego, Calif.). Data shown represent the
fold change in LDH activity relative to vehicle control for each
day of sampling.
GGT activity was measured according to the manufacturer's protocol
(Sigma Aldrich, St. Louis, Mo.). Tissues were washed twice with
DPBS and lysed in GGT assay buffer in a Precellys.RTM. lysis tube
(Precellys, Rockville, Md.). Lysate was assessed for GGT activity
by comparison to a standard curve integration of absorbance
normalized for volume and duration of incubation period at
37.degree. C. using GraphPad Prism.RTM. software (GraphPad, San
Diego, Calif.). Data shown represent the average GGT activity in
mIU/ml for analysis of GGT function over time, or percent relative
to vehicle for toxicity studies.
To measure TEER, individual 3D PT tissues cultured for 21 d were
removed from the Transwell.RTM. insert and loaded into an Ussing
chamber (Physiologic Instruments, San Diego, Calif.). Studies were
run essentially as previously described (Clarke, 2009). Tissues
were bathed in Krebs bicarbonate ringer solution with glucose (115
mM NaCl, 2.4 mM K.sub.2HPO.sub.4, 0.4 mM KH.sub.2PO.sub.4, 1.2 mM
CaCl.sub.2 dihydrate, 1.2 mM MgCl.sub.2 hexahydrate, 25 mM
NaHCO.sub.3-, 10 mM glucose; all reagents from Sigma-Aldrich, St.
Louis, Mo.) and buffer was continuously bubbled with carbogen gas
(95% O.sub.2/5% CO.sub.2). After correcting the electrode offset
potential and liquid resistance, resistance across the tissues was
measured continuously for 1 h.
For passive permeability (P.sub.app) measurements, tissues were
washed with DPBS three times and equilibrated to assay buffer (DPBS
with 10 mM HEPES pH 7.4) for 10 min at 37.degree. C. Tissues were
then dosed with 250 .mu.M Lucifer yellow (Thermo Fisher, Carlsbad,
Calif.) to the apical compartment and fresh assay buffer in the
basolateral (receiver) compartment. Following incubation for 1 h at
37.degree. C., samples were taken from both the apical and
basolateral compartments. Fluorescence in each sample was measured
on a BMG plate reader with an excitation filter of 490 nm and an
emission filter of 540 nm (BMG Labtech, Cary, N.C.) and normalized
to a standard curve for quantification. Papp was calculated with
equation 1, where V represents the volume of Lucifer yellow
solution, T is the duration of the incubation, D.sub.0 is the
concentration of Lucifer yellow applied to the cells, and A is the
growth area of the Transwell.RTM. insert.
P.sub.app=(V/A.times.D.sub.0))*(.DELTA.D/.DELTA.T) Equation 1:
Example 4
ELISA Assay for Angiotensin-Converting Enzyme (ACE) and Angiotensin
II
ACE protein levels in both tissue lysates and conditioned media
were detected by ELISA using the manufacturer's instructions
(Abeam, Cambridge, Mass.). Plates were read at 450 nM (BMG Labtech,
Cary, N.C.) within 30 minutes of addition of the stop solution.
Concentrations of the test samples were determined by comparison to
the standard curve using GraphPad Prism.RTM. software (GraphPad,
San Diego, Calif.).
To evaluate ACE enzyme function, 3D PT tissues were treated for 24
h with 5 ng/ml human angiotensin I (Abeam, Cambridge, Mass.) and
angiotensin II was then detected using a competitive ELISA kit from
Sigma per the manufacturer's instructions (Sigma-Aldrich, St.
Louis, Mo.). Plates were read at 450 nM within 30 minutes of
addition of the stop solution (BMG Labtech, Cary, N.C.). The
concentration of angiotensin II in the test samples was determined
by comparison to the standard curve using GraphPad Prism.RTM.
software (GraphPad, San Diego, Calif.).
Example 5
A Three-Dimensional Renal Tubule Model Bioprinted with Different
Ratios of Renal Fibroblasts to Endothelial Cells
Experiments were undertaken to determine the effect of fibroblast
to endothelial cell ratio on tissue morphology. Renal tubule models
were bioprinted using bio-inks comprising renal fibroblasts and
HUVEC cells at ratios of 90: 10; 75:25; and 50:50 (fibroblast to
endothelial cells).
Example 6
Histology
3D PT were fixed overnight in 2% paraformaldehyde (Electron
Microscopy Sciences, Hatfield, Pa.). Tissues were oriented for
transverse sectioning by pre-embedding in HistoGel.TM. (Thermo
Fisher, Carlsbad, Calif.) and were then dehydrated and infiltrated
with paraffin by automated processing on a Tissue-Tek VIP.RTM.
tissue processing system (Sakura Finetek USA, Torrance, Calif.).
Tissues were sectioned at 5 .mu.M on a Leica.RTM.
Reichert-Jung.RTM. Histocut microtome (Leica Biosystems, Buffalo
Grove, Ill.). Hematoxylin and eosin (H&E) or Gomori's trichrome
(TCM) stains were generated using a Leica.RTM. Autostainer XL
(Leica Biosystems, Buffalo Grove, Ill.) according to manufacturer's
instructions. Immunohistochemistry was performed as previously
described (King et al., 2013) using the primary antibodies in Table
1. Following overnight incubation with primary antibodies at
4.degree. C., sections were stained with AlexaFluor.RTM.-conjugated
secondary antibodies (Thermo Fisher, Carlsbad, Calif.) at 1:200
dilution. For P-gp and SGLT2 detection, tyramide signal
amplification was performed according to the manufacturer's
instructions (Thermo Fisher, Carlsbad, Calif.). Slides were
counterstained and mounted with FluoroGel II with DAPI (Electron
Microscopy Sciences, Hatfield, Pa.). H&E and TCM images were
acquired on a Zeiss.RTM. Axioskop with Zeiss.RTM. Zen software
(Zeiss Microscopy, Thornwood, NY). Immunofluorescent images were
acquired on a Zeiss.RTM. Axio.RTM. Imager A2 with Zeiss.RTM. Zen
software.
TABLE-US-00001 TABLE 1 Dilution Vendor Rabbit .alpha.-CD31 1:100
Abcam (Cambridge, MA) Rabbit .alpha.-TE7 1:500 EMD Millipore
(Temecula, CA) Mouse .alpha.-collagen IV 1:100 Abcam Rabbit
.alpha.-E cadherin 1:50 Abcam Rabbit .alpha.-Pgp 1:500 Abcam Rabbit
.alpha.-SGLT2 1:250 Abcam Rabbit .alpha.-Na.sup.+K.sup.+ATPase
1:100 Abcam Mouse .alpha.-cytokeratin 18 1:500 Abcam Rabbit
.alpha.-PCNA 1:1000 Cell Signaling (Danvers, MA)
Example 7
RNA Isolation and Quantitative RT-PCR
RNA extraction from 3D PT tissues was performed using the Zymo
Direct-zol.TM. RNA kit according to the manufacturer's instructions
(Zymo Research, Irvine, Calif.). RNA was quantified by
spectrophotometry using a NanoDrop.TM. 2000 (Thermo Fisher,
Carlsbad, Calif.) and converted to cDNA using Superscript.RTM. III
First-Strand Synthesis SuperMix according to the manufacturer's
instructions (Thermo Fisher, Carlsbad, Calif.). Amplification
reactions were performed with 200 ng of cDNA using TaqMan.RTM. Gene
Expression Array Cards (Thermo Fisher, Carlsbad, Calif.) with GAPDH
amplification as an endogenous housekeeping control gene.
TaqMan.RTM. probe/primer sets are described in Table 2.
Amplification was detected on a ViiA.TM. 7 real-time PCR system
(Thermo Fisher, Carlsbad, Calif.). Duplicate samples from
individual tissues were assessed. Relative quantitation (RQ) values
for the gene of interest compared to GAPDH were calculated using
the formula RQ=(2-.DELTA..sup.Ct)* 10000. RQ values for each sample
were normalized for KRT18 by dividing the RQ for the gene of
interest compared to GAPDH by the RQ for KRT18 compared to GAPDH.
The fold change was then calculated by dividing the
KRT18-normalized RQ at the experimental day by the KRT18-normalized
RQ for day 3 (or day 12 for SGLT2).
TABLE-US-00002 TABLE 2 Gene Gene Symbol Assay ID ACE ACE
Hs00174179_ml AGT AGT Hs01586213_ml Renin REN Hs00982555_ml MDR1
(P-gp) ABCB1 Hs00184500_ml BCRP ABCG2 Hs01053790_ml AQP1 AQP1
Hs01028916_ml Cubilin CUBN Hs00153607_ml Megalin LRP2 Hs00189742_ml
OCT2 SLC22A2 Hs01010723_ml OAT1 SLC22A6 Hs00537914_ml OAT3 SLC22A8
Hs00188599_ml MATE1 SLC47A1 Hs00217320_ml MATE2K SLC47A2
Hs00945650_ml SGLT2 SLC5A2 Hs00894642_ml
Example 8
Glucose Uptake Colorimetric Assay
Glucose uptake in 3D PT tissues was detected and quantified
according to the manufacturer's protocol (Abeam, Cambridge, Mass.).
Insulin-Transferrin-Selenium (Gibco, Carlsbad, Calif.) was used to
stimulate glucose uptake and canagliflozin (Santa Cruz Biotech,
Dallas, Tex.) was used to inhibit SGLT2 function. Tissues were
starved overnight in DPBS/HEPES, pH 7.4 prior to assay. Tissues
were then pretreated with 1X insulin or 500 .mu.M canagliflozin for
20 minutes, followed by addition of 1 mM 2-deoxyglucose. Tissues
were washed extensively with PBS and lysed in extraction buffer in
Precellys.RTM. lysis tubes (Precellys, Rockville, Md.).
2-deoxyglucose uptake was measured at OD412 nm on a microplate
reader (BMG Labtech, Cary, N.C.) and results were graphed as fold
change relative to control using GraphPad Prism.RTM. software
(GraphPad, San Diego).
Example 9
Vectorial Transport of Rhodamine 123
3D PT tissues were washed with DPBS three times and equilibrated to
assay buffer (DPBS supplemented with 10 mM HEPES, pH 7.4) for 10
min at 37.degree. C. Both apical and basolateral sides of tissues
were then pre-incubated for 20 min at 37.degree. C. in assay buffer
in the presence or absence of 5 .mu.M zosuquidar (Sigma Aldrich,
St. Louis, Mo.). Following pre-treatment, tissues were dosed on the
basolateral side with 1 .mu.M rhodamine 123 (Molecular Probes,
Eugene, Oreg.) with or without 5 .mu.M zosuquidar for 2h at
37.degree. C. After incubation, the tissues were washed with cold
assay buffer, fixed with 2% PFA, and cryosectioned. Images were
captured at the same exposure time across all conditions.
Fluorescence intensity, corrected for background and relative area,
was calculated in Image J (National Institutes of Health, Bethesda,
Md.) and graphed as fold change relative to control using GraphPad
Prism.RTM. software (GraphPad, San Diego).
Statistics were calculated using GraphPad Prism.RTM. software (La
Jolla, Calif.). Data shown is the mean.+-.SEM. Statistical
significance (P<0.05) was calculated by t-test with Dunnet's
post-test, one-way ANOVA, or two-way ANOVA as appropriate.
Example 10
Results-Development and Characterization of a 3D Model of the
Tubulointerstitial Interface of the Human PT
Cultured primary human RPTECs have a finite lifespan in culture
before undergoing epithelial-to-mesenchymal transition or
senescence, with accompanying loss of morphology and function
(Wieser et al., 2008). Abundant evidence supports the notion that
an appropriate microenvironment, including 3D architecture and
supporting cell types, can help maintain and support the continued
health and function of polarized epithelia (Kunz-Schughart et al.,
2006; Bryant and Mostov, 2008; Nagle et al., 2011; Li et al.,
2014). To develop a 3D human system for studying nephrotoxicity,
the Organovo NovoGen Bioprinter.RTM. system was used to create a
model of the PT tubulointerstitial interface (NovoView.TM.
tissues). As shown in the schematic of FIG. 1A, tissues were
designed with a basal multicellular interstitial layer composed of
primary human renal fibroblasts and HUVEC, and an apical monolayer
of polarized primary human RPTEC supported by a basement membrane.
Use of the bioprinter allowed reproducible generation of
spatially-defined tissues created on standard multi-well
Transwell.RTM. inserts (FIG. 1B).
Following culture for 14 days, the PT tissues were analyzed for
tissue organization, cell morphology, and retention of endothelial
and epithelial markers. A hematoxylin and eosin (H&E) stain of
the 3D tissues showed an interstitial layer with low cell density
composed of spindle-shaped fibroblasts and areas of HUVEC
undergoing remodeling to form endothelial cell-lined networks (FIG.
2A). A monolayer of RPTEC cells was observed immediately above the
interstitium, with columnar morphology and basally oriented nuclei.
The interstitial cells themselves secreted abundant ECM as shown by
Gomori's trichrome stain, with fibrillar structures visible
surrounding the endothelial cell networks (red) in the middle of
the tissue as well as underlying the epithelial layer (FIG. 2B).
The putative endothelial cell networks observed by H&E and
trichrome expressed CD31 and demonstrated that the HUVEC had
organized to form open spaces lined by endothelial cells (FIG. 2C).
Separating the interstitium from the epithelium was a collagen
IV-rich basement membrane immediately adjacent to the basal side of
the epithelial cells (FIG. 2D). The RPTEC cells in the 3D PT model
expressed cytokeratin 18 uniformly across the monolayer (FIG. 2E)
with E-cadherin localized laterally between adjacent cells (FIG.
2E). Polarized distribution of Na+/K+ ATPase to the basolateral
membrane of RPTEC was also observed (FIG. 2F).
To assess global tissue metabolic activity and longevity, tissues
were assessed for the ability to reduce resazurin over 4 weeks in
culture in an alamarBlue assay (FIG. 3A). Following an initial
decrease in metabolic activity from day 3 to day 7, the metabolic
activity of the 3D PT tissues continued to increase between day 10
and day 30. To more specifically assess RPTEC functional longevity,
3D PT tissues or tissues lacking epithelium (interstitium only)
were assessed for GGT activity as a function of time in culture.
The 3D PT tissues exhibited an increase in GGT activity from 5
mIU/ml at day 7 to 30 mIU/ml at day 30 (FIG. 3B). As expected, the
interstitium-only tissues lacking RPTEC exhibited negligible GGT
activity throughout the culture period of 30 days (FIG. 3B).
Together, these findings demonstrate the formation of a robust 3D
model of the renal tubulointerstitial interface capable of
supporting RPTEC morphology, viability and function for at least 4
weeks.
Example 11
Characterization of Barrier Function
To measure the barrier function of the 3D PT tissues,
trans-epithelial electrical resistance (TEER) measurements were
performed using an Ussing chamber after 21 days in culture. Table 3
shows the average area-corrected resistance values for 3D PT
tissues, which averaged 18.1 .OMEGA.*cm.sup.2. Passive permeability
(Papp) was also measured in 3D PT tissues by addition of Lucifer
yellow to the apical or basolateral compartment of the
Transwell.RTM. and detection of the fluorophore in the opposite
compartment as a function of time. 3D PT tissues exhibited an
average Papp value of 2.97x10' cm/s indicating that the 3D PT
tissues exhibited a more permeable barrier than observed for
epithelial monolayers with Lucifer yellow (Tran et al., 2004).
Taken together, these values demonstrate that the barrier formed by
the RPTEC cells in the 3D PT tissues is leakier than isolated
epithelial monolayers but characteristic of the barrier observed
for the PT in vivo (Boulpaep and Seely, 1971; Liang et al.,
1999)
TABLE-US-00003 TABLE 3 R (Avg) Papp Tissue .OMEGA.*cm.sup.2
.times.10.sup.-6 cm/s 1 18.1 2.63 2 16.3 2.73 3 13.9 2.66 4 17.4
3.74 5 19.2 3.11 6 23.4 2.93 Avg 18.1 .+-. 1.3 2.97 .+-. 0.17
Example 12
Assessment of the Intrarenal Renin-Angiotensin System (RAS)
To determine whether the 3D PT tissues retained a viable RAS, gene
expression of several members of the pathway were first measured.
Gene expression analysis of the tissues over 30 days in culture
showed detectable levels of ACE, angiotensinogen (AGT), angiotensin
receptor I (AGTR1), and renin (Tables 1 and 4). Consistent with the
gene expression data, ACE protein was detected in both conditioned
media and tissue lysates, with higher detection in the tissue
lysates (FIG. 4A). This may correlate with the observed expression
of ACE in the brush border of the PT (Kobori et al., 2007). To
evaluate the function of ACE, 3D PT tissues were dosed with 5 ng/ml
human angiotensin I for 24 h and assessed for the ability to
convert angiotensin Ito angiotensin II. Following stimulation,
angiotensin II was detected in 3D PT tissues at 0.4 pg/ml (FIG.
4B). Thus the 3D PT tissues exhibited physiologically relevant
features of the in vivo PT, including development of barrier
functions and conversion of angiotensin Ito angiotensin II.
TABLE-US-00004 TABLE 4 Day 12 Day 18 Day 24 Day 30 Day 3 Fold Fold
Fold Fold KRT18- KRT18- change KRT18- change KRT18- change KRT18-
change normalized normalized relative normalized relative
normalized relative no- rmalized relative Target Name RQ RQ to day
3 RQ to day 3 RQ to day 3 RQ to day 3 ACE 10.81 3.84 0.36 6.96 0.64
6.65 0.61 5.75 0.53 AGT 5.69 15.83 2.78 25.03 4.4 25.60 4.5 37.96
6.68 REN 1.18 0.6 0.51 1.84 1.56 2.37 2.02 1.6 1.36
Example 13
Analysis of Renal Transporters in 3D PT Tissues
A key feature of the PT that relates to its susceptibility to
nephrotoxicity is the expression and function of renal
transporters, which take up or efflux compounds from the
capillaries surrounding the PT or the glomerular filtrate in the
lumen of the tubule. Primary human RPTEC dedifferentiate rapidly
when cultured in 2D, exhibiting varying levels of renal
transporters and a range of cellular morphologies depending on the
time and method of culture (FIGS. 11A-11E and Wieser et al., 2008;
Vesey et al., 2009). We hypothesized that culturing low-passage
primary human RPTEC on a relevant renal interstitium would preserve
transporter expression and function. To validate the use of the 3D
PT model for transporter-dependent toxicity studies, tissues were
first analyzed for relative expression levels of key renal
transporter genes by qPCR (Tables 1 and 5). For the individual
donor cells incorporated into 3D PT tissues in this study, nearly
all renal transporters evaluated exhibited stable, detectable gene
expression for greater than 4 weeks in culture (Table 5). The
xenobiotic transporter OCT2 was detected at relatively high levels
throughout 28 days in culture, with a 1.8-fold increase in
expression at day 30 compared to day 3 (Tables 1 and 5). Luminal
reabsorption transporters for endogenous substrates, including
cubilin, megalin, AQP1, and SGLT2 exhibited detectable expression
over 30 days in culture. Megalin expression decreased slightly
after 18 days in culture, but SGLT2 expression increased over time
(Tables 1 and 5). Of the efflux transporters analyzed, P-gp
exhibited the highest expression level, with peak expression
between days 18 and 30 in culture (Tables 1 and 5).
TABLE-US-00005 TABLE 5 Day 12 Day 18 Day 24 Day 30 Day 3 Fold Fold
Fold Fold KRT18- KRT18- change KRT18- change KRT18- change KRT18-
change normalized normalized relative normalized relative
normalized relative no- rmalized relative Target Name RQ RQ to day
3 RQ to day 3 RQ to day 3 RQ to day 3 ABCB1 2.77 18.91 6.82 63.79
23.02 42.18 15.22 77.6 28 (MDR1, P-gp) ABCG2 9.82 1.58 0.16 15.94
1.62 2.77 0.28 0.84 0.09 (BCRP) AQP1 5.42 4.89 0.9 5.44 1 4.72 0.87
8.21 1.52 CUBN 0.63 10.74 17 15.03 23.79 11.29 17.88 21.13 33.46
LRP2 0.83 0.69 0.83 2.07 2.5 0.92 1.12 0.54 0.66 (megalin) SLC22A2
8 14.71 1.84 22.09 2.76 6.91 0.86 14.45 1.81 (OCT2) SLC22A6 ND ND
ND ND ND ND ND ND ND (OAT1) SLC22A8 ND ND ND ND ND ND ND ND ND
(OAT3) SLC47A1 3.78 3.52 0.93 4.85 1.28 1.87 0.5 1.55 0.41 (MATE1)
SLC47A2 0.79 4.69 5.98 2.15 2.74 2.31 2.94 4.88 6.21 (MATE2K)
SLC5A2 ND 0.88 ND 0.63 0.71 2.03 2.3 3.02 3.43 (SGLT2)
(OAT3), SLC47A1 (MATE1), SLC47A2 (MATE2K), and SLC5A2 (SGLT2)
expression relative to GAPDH and normalized to KRT18 between days 3
and 30 of culture. Data shown is the average relative
quantification (RQ) compared to GAPDH and normalized to KRT18 and
the fold change compared to day 3 for 2 tissues per time point. For
SGLT2, fold change shown is relative to day 12. ND, not
detected.
To further assess both uptake and efflux transporter function in
the 3D PT model, the glucose uptake transporter SGLT2 and the
xenobiotic efflux transporter P-gp were selected for functional
analysis. As shown in FIG. 5A, SGLT2 protein expression was
detected primarily at the apical surface of RPTEC on 3D PT tissues
(3D tissues were stained with antibodies against SGLT2). This
pattern matches what is seen in vivo in the human PT (Brenner,
2008). To evaluate SGLT2 transporter function, tissues were kept in
either normal tissue maintenance media or starved of glucose for 24
h, followed by stimulation of glucose uptake by insulin in the
presence or absence of the SGLT2 transport inhibitor canagliflozin
(FIG. 5B). In tissues maintained in normal tissue media, treatment
with insulin induced a 4-fold increase in intracellular 2-DG, which
decreased by 50% upon co-administration of the SGLT2 inhibitor
canagliflozin (FIG. 5B, black and grey bars). This suggests that
there is functional SGLT2 transport in the tissues, and that other
transport mechanisms are also contributing to global glucose
uptake. When tissues were starved overnight, insulin induced an
8-fold increase in glucose uptake, which was significantly reduced
by canagliflozin to levels indistinguishable from the control
tissues (FIG. 5B). As expected, starvation increased glucose uptake
by 3D PT tissues beyond that observed for tissues cultured in
normal media as the tissues sought to re-establish glucose
homeostasis lost during culture in the absence of glucose, and
SGLT2 appears to play a role in this process.
To assess P-gp mediated efflux capabilities in 3D PT tissues, we
first wanted to determine the localization of the transporter
protein. As expected for native proximal tubule, P-gp protein
expression was detected at the apical surface of the RPTEC cells in
the 3D PT model. After 14 days in culture, 3D PT tissues were
stained with antibodies against P-gp (FIG. 6A and (Brenner, 2008)).
To evaluate P-gp function, 3D PT tissues were loaded with rhodamine
123 (R123) in the presence or absence of zosuquidar, a P-gp
inhibitor. Following uptake, tissues were washed and cryosectioned
to detect the presence of R123 in the RPTEC of the PT model.
Tissues treated with buffer alone exhibited no green fluorescence
(control), while tissues treated with R123 exhibited punctate
fluorescent expression in the cytoplasm of the RPTEC. Upon blocking
P-gp- mediated efflux with zosuquidar, an increase in accumulated
fluorescence was observed in the epithelium with the RPTEC
monolayer fluorescing uniformly throughout the cytoplasm (FIG. 6B).
Image quantification showed that tissues exposed to R123 exhibited
a 4-fold increase over control tissues, while treatment with R123
plus zosuquidar resulted in a 6-fold increase in fluorescence over
control tissues and a significant increase compared to R123-
treatment alone (FIG. 6C). Thus the 3D PT tissues exhibited stable
expression of renal transporters over time, and functional activity
of the endogenous substrate transporter SGLT2 and the xenobiotic
transporter P-gp were verified.
Example 14
Assessment of Cisplatin Nephrotoxicity Using 3D PT Tissues
Cisplatin is a chemotherapeutic agent with multiple mechanisms of
action that lead to nephrotoxicity, including generation of
reactive oxygen species and formation of toxic glutathione
conjugates following concentration of the molecule in RPTEC by
renal uptake transporters including OCT2 (Hanigan and Devaraj an,
2003; Yonezawa et al., 2005). In addition, cisplatin has been
reported to lead to tubulointerstitial fibrosis (Guinee et al.,
1993). To assess whether the 3D PT tissues could manifest cisplatin
toxicity, tissues were exposed daily to cisplatin followed by
measurement of overall viability, levels of GGT activity, release
of LDH, and histological analysis. Tissues treated with cisplatin
exhibited a significant decrease in alamarBlue.TM. metabolism at
doses as low as 1 with an LD 50 value of 5.72 .mu.M and complete
loss of viability at 10 .mu.M (FIG. 7A). A similar pattern was
observed for GGT activity in response to cisplatin, with 5 .mu.M
cisplatin causing a nearly 50% reduction in GGT activity,
indicating a significant effect on the RPTEC of the 3D PT model
(FIG. 7B).
To evaluate the role of OCT2-mediated transport in the
cisplatin-induced toxicity, tissues were treated with cisplatin in
the presence of cimetidine, an OCT2 inhibitor. As shown in FIG. 8A,
no loss of viability was observed in tissues treated with
cimetidine alone compared to the vehicle control. Tissues treated
with 5 .mu.M cisplatin exhibited a nearly 50% decrease in viability
(FIG. 7A and FIG. 8A), and tissues treated with a combination of
cisplatin and cimetidine exhibited viability levels
indistinguishable from vehicle or cimetidine-only controls. This
protective effect of cimetidine was also observed in the GGT
activity levels from tissues treated with cisplatin or cisplatin
plus cimetidine (FIG. 8B). LDH release, indicative of toxicity,
peaked at treatment day 5 in tissues treated with 5 .mu.M cisplatin
alone, with an observed 3-fold increase over vehicle controls (FIG.
8C). Tissues treated with cisplatin plus cimetidine did not exhibit
the same damage response, showing only slightly elevated levels of
LDH release compared to vehicle at day 5 and indistinguishable
levels versus control-treated tissues by day 7 (FIG. 8C).
Histological analysis by H&E staining confirmed the loss of
epithelial viability in response to cisplatin (FIGS. 9A-9D).
Vehicle or cimetidine-only tissues exhibited healthy, columnar
RPTEC with round nuclei (FIGS. 9A and B), while tissues treated
with 5 .mu.M cisplatin exhibited a more squamous morphology and
loss of nuclei (FIG. 9C). Tissues treated with cisplatin plus
cimetidine exhibited a substantial improvement in epithelial
morphology versus cisplatin alone, with partial restoration of
nuclear localization and columnar RPTEC (FIG. 9D). To evaluate
RPTEC proliferation in response to damage induced by cisplatin,
tissues were stained for proliferating cell nuclear antigen (PCNA).
Vehicle or cimetidine-treated control tissues exhibited low levels
of RPTEC proliferation; however, a dose-dependent proliferative
response was observed in tissues treated with cisplatin (FIGS.
10A-10D). This increased proliferation in the RPTEC of 3D PT
tissues was decreased by co-administration of cimetidine. Thus, the
3D PT tissues were able to recapitulate nephrotoxicity after
exposure to clinically-relevant doses of cisplatin and confirm the
role of the OCT2 transporter as a mechanism of nephrotoxicity
induction.
Example 15
Assessment of Complications of Diabetes
ExVive.TM. Human Kidney Tissue (Organovo, San Diego, Calif.) is a
fully human 3D bioprinted tissue comprised of an apical layer of
polarized primary renal proximal tubule epithelial cells (RPTECs)
supported by a collagen IV-rich tubulointerstitial interface of
primary renal fibroblasts and endothelial cells. After culturing
for 14 days, Healthy ExVive.TM. kidney tissues were either
untreated (control) or were exposed to a high concentration of
glucose (1000 mg/dL=10 g/L) for an additional 14 days to mimic the
high levels of urine glucose seen in diabetic patients.
The tissues were then formalin fixed, embedded, sectioned, and
stained with hemotoxylin and eosin (H&E) to look for changes in
cellular and nuclear morphology. As shown in FIGS. 12B and 12D,
high glucose treatment leads to the generation of glycogenated
nuclei in the epithelial cells layer (as shown by the arrows). The
glycogenated nuclei are characterized by nuclei with a "hollowed
out" appearance due to the storage of glycogen, with condensation
of chromatin around the nuclear membrane and the presence of a
prominent nucleolus (see insert in FIG. 12D). The presence of
glycogenated nuclei has been observed in diabetic patient and
rodent models.
Example 15 shows that an isolated, 3D printed proximal tubule
disorder model can be successfully induced to exhibit the desired
phenotype without the proximal tubule's usual support system
inducing this phenotype. The usual support system of the proximal
tubules would include, by way of example, the glomeruli, the
Bowman's capsule, and the surrounding perfusion system for the
proximal tubules. In a diabetic patient, high concentrations of
glucose may accumulate in the glomeruli, which may gradually
contribute to the dysfunction of the glomeruli. This gradual
dysfunction of the glomeruli eventually results in a leakage of
excess glucose into the proximal tubules. As such, the proximal
tubules may begin to develop an acute or chronic diabetic disorder
displaying the presence of a glycogenated nuclei. Example 15 also
displays the presence of glycogenated nuclei, which shows that an
isolated, 3D bioprinted proximal tubule disorder model can be
successfully induced to exhibit the desired disorder phenotype
without the proximal tubule's usual support system inducing this
phenotype.
Example 16
Assessment of Crystalline Deposits
Healthy ExVive.TM. kidney tissues were either untreated (control)
or exposed to a nephrotoxic agent.
The tissues were then formalin fixed, embedded, sectioned, and then
stained to look for calcium oxalate deposits. Calcium oxalate is a
known component of kidney stones. As shown in FIGS. 13B and 13C,
exposure of the kidney tissues to a nephrotoxic agent produces
treatment-dependent deposits (as shown by the arrows).
Example 17
Renal Fibrosis--ExVive.TM. Human Kidney Tissue Treated with
TGF.beta.
As described in this present disclosure, the ExVive.TM. Human
Kidney Tissue (Organovo, San Diego, Calif.) is a fully human
three-dimensional (3D), bioprinted tissue comprised of an apical
layer of polarized primary renal proximal tubule epithelial cells
(RPTECs) supported by a collagen IV-rich tubulointerstitial
interface of primary renal fibroblasts and endothelial cells. As
renal fibrosis is a common downstream effect of drug-induced
injury, the ExVive.TM. Human Kidney Tissue was treated with
Transforming Growth Factor .sub.R (TGFI3 or TGFbeta), a key player
of the fibrotic response, for purposes of evaluating
tubulointerstitial fibrosis. Thus, to induce renal fibrosis, a
healthy ExVive.TM. Human Kidney Tissue was treated with TGFI3, a
key player of the fibrotic response. In particular, each healthy
ExVive.TM. Human Kidney Tissue was dosed daily for seven days with
vehicle control, 0.37 ng/ml TGF.beta., 1.1ng/m1 TGF.beta., 3.3
ng/ml TGF.beta., or 10 ng/m1 TGF.beta., respectively. Subsequently,
after the ExVive.TM. Human Kidney Tissue was treated with
TGF.beta., an assessment of the viability and epithelial cell
functions of this ExVive.TM. Human Kidney Tissue was conducted.
FIG. 14A shows the analysis of Resazurin conversion as measure of
overall tissue metabolic activity and cell health. No statistically
significant differences were detected between treatment groups.
FIG. 14B shows the analysis of gamma glutamyl transfer (GGT)
activity as a measure of epithelial cell function. Statistically
significant reduction in RPTEC function was detected with higher
doses of TGF.beta.. Data shown is the average of 3 tissue samples
per condition and is represented as the fold change relative to the
vehicle control. *p<0.05; ***p<0.001 for each condition
compared to vehicle control.
Fibrosis-related gene expression was also assessed on this
ExVive.TM. Human Kidney Tissue treated with TGF.beta.. FIG. 15
shows gene expression analysis by semi-quantitative RTPCR showed
induction of the fibrotic markers collagen I (COL1A1), connective
tissue growth factor (CTGF), fibroblast-activating protein (FAP),
or platelet-derived growth factor receptor beta (PDGFRB). Data
shown is the average of 3 tissue samples per condition.
*p<0.0001 for each condition compared to vehicle control.
In the ExVive.TM. Human Kidney Tissue treated with TGF.beta., it
was also shown that TGF.beta. induces tissue thickening and
increased extracellular matrix deposition. FIG. 16A shows
representative Gomori's Trichrome stains for ECM deposition. As
shown in FIG. 16A, increased TGF.beta. induced an increase in the
extracellular matrix deposition. FIG. 16B shows quantification of
Sirius red-stained collagen in tissue sections. Data represents the
average of 4 technical replicates per tissue, 3 tissues per
condition and is represented as the fold change relative to the
vehicle control following normalization to total protein content as
measured by Fast Green staining. *p<0.05 for each condition
compared to vehicle control.
Example 17 (FIGS. 14-16) show that renal interstitial fibrosis can
be induced in the ExVive.TM. Human Kidney Tissue by treating with
TGF.beta.. Following treatment of ExVive.TM. Human Kidney Tissue
with TGF.beta. for 7 days, an increase in the expression of
fibrosis-related genes (FIG. 15), extensive ECM deposition in the
interstitium (FIG. 16A), and loss of epithelial cell function at
the highest dose (FIG. 14B) were shown. This is a significant
technical advancement in the state-of-the-art because these results
demonstrate the extended capabilities of the ExVive.TM. Human
Kidney Tissue to mount measurable responses at the biochemical,
transcriptional, and histological levels consistent with renal
injury and interstitial fibrosis, a disease phenotype not
achievable in traditional systems of epithelial cell monolayer
culture with limited longevity. The ExVive.TM. Human Kidney Tissue
could therefore enable applications aimed at understanding
mechanisms of disease progression, evaluating drug-induced renal
fibrosis, and investigation of intervention strategies toward the
development of novel anti-fibrotic drugs.
Example 18
ExVive.TM. Human Kidney Tissue Treated with Cisplatin
Cisplatin was used to induce nephrotoxicity. As previously
described in FIGS. 7-10, cisplatin can induce nephrotoxicity. Each
healthy ExVive.TM. Human Kidney Tissue was dosed daily with 5, 10,
or 25 .mu.M cisplatin, respectively. As described in this present
disclosure, the ExVive.TM. Human Kidney Tissue (Organovo, San
Diego, Calif.) is a fully human three-dimensional (3D), bioprinted
tissue comprised of an apical layer of polarized primary renal
proximal tubule epithelial cells (RPTECs) supported by a collagen
IV-rich tubulointerstitial interface of primary renal fibroblasts
and endothelial cells.
Following treatments (Tx) 3 and 5, media supernatants were
collected and analyzed for cytokeratin 18 fragments M30 and M65, as
shown in FIG. 17. As shown in FIG. 17, there was increased soluble
CK18 following cisplatin treatment of the ExVive.TM. Human Kidney
Tissue. Cytokeratin 18 (CK18) is a surface marker produced in
epithelial cells. Secreted cleavage products from CK18 differ based
on the mechanism of cell death and can be measured from spent media
samples with solid-phase sandwich enzyme ELISA (Diapharma, West
Chester, Ohio). The M30 CK18 fragment is produced by enzymatic
caspase activity stemming from apoptosis, while the M65 CK18
fragment is released from dead cells (apoptotic and necrotic). This
ELISA assay provides the ability to distinguish the magnitude of an
epithelial specific injury within in tissue comprised of multiple
cell types. In tandem, M30 and M65 helps to determine mechanism of
epithelial cell death.
Example 19
Detection of Transporter Protein Expression in ExVive.TM. Human
Kidney Tissue
FIGS. 18A-I show human renal cortex samples (KT1 and KT2),
ExVive.TM. Human
Kidney Tissue (3D-1 and 3D-2), and plated 2D RPTEC cells (2D RPTEC
lot 1105) were analyzed for transporter expression by LC-MS/MS. As
described in this present disclosure, the ExVive.TM. Human Kidney
Tissue (Organovo, San Diego, Calif.) is a fully human
three-dimensional (3D), bioprinted tissue comprised of an apical
layer of polarized primary renal proximal tubule epithelial cells
(RPTECs) supported by a collagen IV-rich tubulointerstitial
interface of primary renal fibroblasts and endothelial cells.
Peptides unique for each transporter were selected based on in
silico selection criteria. Total membrane isolation was performed
on tissue/cell samples prior to analysis. Each bar represents
transporter peptide peak area normalized to total sample protein
and to spiked human serum albumin internal standard. Note that
FIGS. 18A-I describe protein expression of transporters, wheras
FIG. 11 describes gene expression of transporters.
All transporters analyzed were detectable in renal cortex samples,
ExVive.TM. Human Kidney Tissue, and plated 2D RPTECs. The
transporters measured are critical for drug disposition within the
human kidney. As a result, these transporters have been identified
by regulatory approval bodies, such as the United States Food and
Drug Administration (FDA) and the European Medicines Agency (EMA)
as critical transporters to evaluate for drug safety in the human
kidney.
Discussion
To date, very few systems have been developed to study the human
renal tubulointerstitial interface in vitro. A variety of systems
for 3D culture of RPTEC in isolation have been developed, including
culturing cells in Matrigel.RTM., culturing cells as organoids on a
variety of scaffolds such as hyaluronic acid or silk, and culture
of RPTEC in microfluidic devices ("kidney on a chip") (Joraku et
al., 2009; Subramanian et al., 2010; Astashkina et al., 2012; Jang
et al., 2013). However, these systems lack direct contact between
the epithelium and relevant interstitial cell types, including
fibroblasts and endothelial cells, that play both a structural role
in orienting the epithelium as well as providing a source of growth
factors critical for the continued health and organization of the
epithelium (Lemley and Kriz, 1991; Kaissling and Le Hir, 2008;
Meran and Steadman, 2011). Without these supportive cell types,
RPTEC rapidly lose their native phenotype in culture, thus
preventing the ability to perform the chronic, low dose exposure
studies necessary to predict how a molecule will perform in the
clinic. The goal of this study was to use 3D bioprinting to build
and characterize a model in which a renal interstitium supported
the continued growth and maintenance of healthy epithelia. The
renal fibroblasts and endothelial cells provided a robust source of
endogenously-produced extracellular matrix, which enabled tissue
formation without the use of exogenous scaffolding as well as
supported the formation of open networks of endothelial cells in
the interstitial layer and a collagen-rich basement membrane
underlying the epithelium. The endothelial networks form open
spaces in the interstitium that may allow better access of media
and nutrients to the entirety of the tissue. While the interstitial
layer is thicker than the native human renal interstitium, the
combination of the renal fibroblasts with the endothelial cells
does enable a cellular density more reminiscent of the in vivo
tissue, which contains fibroblast-like cells immediately adjacent
to the epithelium (Lemley and Kriz, 1991).
3D PT tissues were evaluated for their ability to recapitulate
physiologically-relevant aspects of the in vivo proximal tubule,
including reconstitution of the intrarenal RAS and barrier
functions. The human PT expresses ACE at the apical surface of
RPTEC in order to convert angiotensin Ito angiotensin II (Schulz et
al., 1988; Ichihara et al., 2004), which then plays a critical role
in regulating sodium transport to influence hypertension through
feedback onto the renal microvasculature and glomerulus (Kobori et
al., 2007). The 3D PT model was able to demonstrate angiotensin II
conversion in response to angiotensin I stimulation (FIG. 4B).
Future experiments exploring the RAS in the 3D PT model could
potentially be used to separate the effects of new therapeutics for
hypertension on the glomerulus versus the PT, particularly with
regard to mitigating nephrotoxicity as a result of hypertension.
Another important function of the PT is to serve as an epithelial
barrier controlling the movement of specific types of molecules
across the monolayer. The PT is the primary site of re-uptake of
water and solutes following glomerular filtration, and as such,
must form a more permeable barrier than that observed more distally
in the nephron (Ussing et al., 1974; Greger, 1996). Monolayer
cultures of renal epithelial such as LLC-PK1 and MDCK cells have
been shown to exhibit higher TEER values of 100-200 .OMEGA.*cm2,
while in vivo tubules exhibit values between 6.6 and 11.6
.OMEGA.*cm2 (Boulpaep and Seely, 1971; Liang et al., 1999). In this
study, 3D PT tissues exhibited TEER values of 18.1 .OMEGA.*cm2
(Table 3), which more closely matches values measured in vivo for
PT barrier formation. Monolayer epithelial cultures with tight
barrier function and high TEER values (>100 .OMEGA.*cm2) exhibit
a Papp of 0.5-1.times.10.sup.-6 cm/s for Lucifer yellow (Tran et
al., 2004). The average Papp value for Lucifer yellow in 3D PT
tissues was 2.97.times.10.sup.-6 cm/s, indicating transcellular or
paracellular transport through the tissues and confirming the leaky
barrier function noted by TEER measurements (Table 3). One possible
cause for this is the presence of the extracellular matrix-rich
interstitium underlying the RPTEC, which may support the formation
of a leaky barrier through formation of a physiologically-relevant
basement membrane structure.
Primary human RPTEC provide the advantage of expressing a variety
of transporters known to play a role in drug-induced kidney injury;
however, these cells can be cultured for a limited time (<14
days) before undergoing senescence or epithelial to mesenchymal
transition and concomitant loss of renal transporter expression and
function (FIGS. 11A-11E). In contrast, culturing these cells in a
3D context on an interstitial layer enabled retention of epithelial
cell viability and function for at least 30 days in culture while
retaining gene expression of many renal transporters such as
cubilin and megalin, MATE1 and MATE2K, OCT2, BCRP, and P-gp (Tables
1 and 5). Furthermore, polarized distribution and functional
activity of P-gp and SGLT2 were confirmed in 3D PT tissues by
transport of a glucose analog and R123, respectively (FIGS. 5A-B
and FIGS. 6A-6E). The continued expression and function of renal
transporters in the 3D PT tissues allows the possibility of
performing chronic dosing studies to assess human nephrotoxicity
coupled with detailed analysis of molecular mechanisms of
action.
A human 3D multi-cellular renal tissue composed of distinct
epithelial and interstitial cell compartments provides a unique
test platform for evaluating new drug entities for potential
nephrotoxicity, allowing for the assessment of biochemical,
transcriptional, and histological endpoints across multiple cell
types and anatomical locations ex vivo. To provide initial
proof-of-concept data that this model may be used for
nephrotoxicity testing, 3D PT tissues were exposed to the classical
nephrotoxin cisplatin. 3D PT tissues exhibited an LD50 value of
5.72 .mu.M (FIG. 7A), consistent with previously reported values
for in vitro and ex vivo cisplatin toxicity (Tay et al., 1988;
Katsuda et al., 2010). While several mechanisms likely play a role
in cisplatin-mediated nephrotoxicity, including generation of
reactive oxygen species and creation of toxic intermediates through
glutathione conjugation, these mechanisms occur after cisplatin has
been taken up by RPTECs (Hanigan and Devarajan, 2003). This uptake
is thought to occur primarily through the action of the OCT2 renal
transporter, although other transporters such as the copper
transporters (CTR1 and 2) may play a role as well (Ciarimboli et
al., 2005). In the current study, inhibition of the OCT2
transporter by cimetidine successfully protected against
cisplatin-induced loss of viability and epithelial function (FIGS.
8A-8C and FIGS. 9A-9D). This mechanism is clinically relevant, as
polymorphisms in OCT2 that influence its function are predictive of
cisplatin-induced AKI, and animal models that lack OCT2 expression
exhibit decreased sensitivity to cisplatin (Ciarimboli et al.,
2005; Ciarimboli et al., 2010). In response to AKI, the PT
epithelium has demonstrated a high capacity for compensatory
proliferation and repopulation in vivo (Nadasdy et al., 1994).
Analogously, we observed a dose-dependent increase in proliferating
RPTEC in 3D PT tissues exposed to cisplatin, which was reduced in
tissues treated with cimetidine (FIGS. 10A-10D). In humans,
cimetidine therapy or the presence of loss-of-function mutations in
OCT2 correlated with decreased urinary cystatin C following
cisplatin administration, demonstrating the possible utility of
this therapy as an ameliorative during chemotherapy (Zhang and
Zhou, 2012).
In summary, we have designed and validated a new in vitro human 3D
tissue model capable of preserving RPTEC function over an extended
time in culture and enabling quantitative detection of PT
nephrotoxicity occurring by specific mechanisms. These data suggest
that 3D PT tissues could positively impact the pre-clinical drug
discovery pipeline, helping to prevent costly failures in late
stage clinical trials. Use of primary human RPTECs from multiple
donors, including those from patients with acute or chronic kidney
disease, may enable better understanding of how drugs may perform
clinically across a specific patient population. Additional studies
across a panel of nephrotoxic compounds with differing mechanisms
of action will help to further elucidate the value of the system
for screening new chemical entities. The inclusion of a
tubulointerstitial interface in the model allows for exploration of
complex, multifactorial disease processes like fibrosis, as well as
assessing the capacity of the RPTEC to repopulate and regenerate
during or after drug-induced injury. In addition, the system may
enable the parallel investigation of biomarkers that may be useful
in noninvasively detecting early kidney injury.
Abbreviations
3D, three-dimensional; ACE, angiotensin-converting enzyme; AGT,
angiotensinogen; AGTR1, angiotensin receptor type I; AKI, acute
kidney injury; DPBS, Dulbecco's phosphate buffered saline; H&E,
hematoxylin and eosin; HUVEC, human umbilical vein endothelial
cell; LDH, lactate dehydrogenase; OCT, organic cation transporter;
Papp, passive permeability; PCNA, proliferating cell nuclear
antigen; PT, proximal tubule; R123, rhodamine 123; RAS,
renin-angiotensin system; RFU, relative fluorescence units; RPTEC,
renal proximal tubule epithelial cell; TEER, trans-epithelial
electrical resistance.
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While preferred embodiments of the present invention have been
shown and described herein, it will be obvious to those skilled in
the art that such embodiments are provided by way of example only.
Numerous variations, changes, and substitutions will now occur to
those skilled in the art without departing from the invention. It
should be understood that various alternatives to the embodiments
of the invention described herein may be employed in practicing the
invention.
All patents, patent applications and publications cited herein are
fully incorporated by reference herein.
* * * * *
References